Cubilin protein, DNA sequences encoding cubilin and uses thereof

ABSTRACT

The present invention provides novel renal receptors for ligands. Cubilin and megalin are representative examples of such renal receptors. Also provided are potential uses of these renal receptors for treating toxicity in various tissues and for detecting renal damage.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is the national stage of PCT/US99/01259 Jan. 21, 1999 claims benefit of provisional patent application U.S. Serial No. 60/072,197, filed Jan. 22, 1998, now abandoned.

FEDERAL FUNDING NOTICE

The present invention was funded in part by NIH grant DK46117. Consequently, the United States government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecular biology, biochemistry and medical therapy. More specifically, the present invention relates to major renal receptors for low molecular weight proteins and potential uses of the receptors for therapy to prevent renal toxicity.

2. Description of the Related Art

Countless proteins of small or intermediate molecular weight, filtered freely or partially through the renal glomerulus, are bound by scavenger pathway receptors on the luminal surface of proximal tubular cells for reuptake (Batuman et al., 1990; Birn et al., 1997; Christensen et al., 1995; Saito et al., 1994). These proteins are then transcytosed back into the circulation, or degraded, releasing amino acids for fresh protein synthesis. The scavenger pathway receptors of the proximal tubular are an essential physiological defense against the urinary loss of a diverse array of plasma proteins essential to homeostatic functions from coagulation to lipid metabolism. Unfortunately, exposure of the scavenger receptors to unusually high concentrations of ligands due to overproduction of a ligand such as myeloma light chains introduction of a freely filtered drug such a s gentamicin, or increased glomerular permeability can disrupt the physiological balance, resulting in severe nephrotoxicity.

The relative contribution of proximal and distal elements to the development of protein nephrotoxicity remains controversial and ill-defined. For light chain nephrotoxicity if they fail to be reabsorbed proximally, the ligands are delivered into the distal tubular segments of the nephron, where they precipitate as casts in combination with Tamm-Horsfall protein (Huang et al., 1997; Weiss et al., 1981; Winearls, 1995), the severity of the renal dysfunctional correlates with the degree of (distal) cast formation (Myatt, 1994; Winearls, 1995). However, some light chains are associated with a pure (proximal) Fanconi syndrome. Myoglobin on the other hand is associated with little (distal) cast formation, but marked proximal tubular damage, with clinical acute tubular necrosis (Paller, 1988; Zager, 1991).

Immunoglobulin light chains are filtered at the glomerulus and endocytosed in the proximal tubule (Batuman et al., 1990; Batuman et al., 1997). In overproduction states, such as multiple myeloma, light chains, also known as Bence-Jones proteins, may produce nephrotoxicity. It was shown previously that free κ- and λ-light chain isotypes bind to a single class of renal proximal tubular receptors which facilitate internalization and degradation (Batuman et al., 1997). To date, however, the receptor(s) which mediate endocytosis of light chains in the proximal tubule have not been characterized.

It has long been postulated that glycoproteins expressed at the apical pole of proximal tubule cells of the kidney acted as scavenger pathway receptors. The only known and cloned receptor until now is megalin, a fairly abundant proximal tubule protein, also known as gp330 or the “Heymann antigen”. Megalin is a classic single transmembrane domain giant glycoprotein receptor (Saito et al., 1994), which belongs to the LDLR family (Yamamoto, 1984), and is closely related to the α₂-macrogilobulin receptor, which is not expressed in the kidney (Moestrup, 1994). Characterization of megalin revealed that, like the α₂M receptor, it was a multiligand receptor. Of particular interest for the renal pathology, megalin binds tPA and urokinase in complex with the corresponding inhibitor, but is also a polybasic drug receptor, binding ligands such as the aminoglycoside antibiotics (Moestrup et al., 1995).

Thus the prior art is deficient in the lack of renal receptors for toxic, physiological, and pathological proteins and drugs (such as myeloma light chains) and more generally, components that may gain access to the proximal tubule fluid. Further, the prior art is deficient in the lack of effective means of preventing renal toxicity by utilizing renal binding proteins or fragments thereof for such components. The present invention fulfills these long-standing needs and desires in the art.

SUMMARY OF THE INVENTION

The present invention discloses renal binding proteins for ligands. Also disclosed are the potential uses of these proteins for therapy to prevent renal toxicity or other types of toxicity.

In one embodiment of the present invention, there is provided a DNA encoding a cubilin protein selected from the group consisting of: (a) isolated DNA which encodes a cubilin protein; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes a cubilin protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a cubilin protein. Preferably, the DNA has the sequence shown in SEQ ID No. 1, and cubilin protein has the amino acid sequence shown in SEQ ID No. 2. Still preferably, the DNA is expressed in the tissues like kidney, spleen, brain, liver, heart and thyroid.

In one embodiment of the present invention, there is a vector capable of expressing the DNA adapted for expression in a recombinant cell and regulatory elements necessary for expression of the DNA in the cell. Specifically, the DNA encodes a cubilin protein.

In another embodiment of the present invention, there is a host cell transfected with the vector expressing a cubilin protein. Specifically, the host cell is selected from the group consisting of bacterial cells, mammalian cells, plant cells and insect cells. More specifically, the bacterial cell is E. coli.

In another embodiment of the present invention, there is provided isolated and purified cubilin protein or fragment coded for by DNA selected from the group consisting of: (a) isolated DNA which encodes a cubilin protein or fragment; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes a cubilin protein or fragment; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a cubilin protein or fragment. Preferably, the protein has the amino acid sequence shown in SEQ ID No. 2, and the fragment has amino acid sequence consisting of one or more of the sequences selected from the group consisting of SEQ ID Nos. 21-27.

In another embodiment of the present invention, there is provided a method of detecting expression of the cubilin protein or fragment in a sample, comprising the steps of: (a) contacting mRNA obtained from the sample with a labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA.

In another preferred embodiment of the present invention, there is provided a pharmaceutical composition comprising the cubilin protein or fragment and a pharmaceutically acceptable carrier. Such composition can be used for treating or reducing nephrotoxicity or other types of toxicity in an in-need individual.

In still another embodiment of the present invention, there is provided a receptor for a variety of ligands, comprising a cluster of EGF repeats and a cluster of CUB domains. Specifically, the receptor is cubilin and the ligand is selected from the group consisting of immunoglobulin light chain, myoglobin, intrinsic factor-vitamin B₁₂, metallothionein, β-2-microglobulin, amyloid, hemoglobin, haptoglobin, interferon, insulin, cytochrome c, lysozyme, transferrin, transthyretin, polybasic drugs, apolipoprotein AI, high density lipoprotein and receptor related protein. More specifically, a representative example of polybasic drug is gentamicin. Representative examples of immunoglobulin light chain include κ-light chain and λ-light chain.

In still yet another embodiment of the present invention, there is provided a method of detecting renal damage by measuring the level of cubilin in the urine of an individual suspected to have such damage. If the urinary cubilin level is lower than that of a normal individual, the test individual might have chronic renal damage. On the other hand, the test individual might have renal damage of acute origin if the urinary cubilin level is higher than that of a normal individual.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

FIG. 1 shows cDNA cloning of the rat yolk sac cubilin. The 11.6 kb cDNA sequence was encoded by three clones (b-d) from a rat yolk sac cell cDNA library. The ends of the cDNA were confirmed by sequencing 3′- and 5′-RACE products from a rat kidney cDNA library with end-ligated adaptors. A polyadenylation signal followed by a poly(A) tail is present 0.4 kb downstream the open reading frame (0.4-11.2 kb). The position of the initially identified clone is indicated (a).

FIG. 2 shows a Northern blot of yolk sac RNA using a cubilin RNA probe.

FIGS. 3A-3C show amino acid sequence of rat cubilin (SEQ ID NO. 2) as deduced by cDNA cloning. The predicted 20 amino acid endoplasmatic import signal sequence is shown in italic type. Potential glycosylation sites are indicated by asterisks. The sequence verified by protein sequencing of tryptic peptides are shown in boldface type. The estimated mass of the peptide backbone is 396,953 kDa and pI is 5.6.

FIG. 4 shows deglycosylation of cubilin purified by IF-B₁₂ affinity chromatography of rabbit renal cortex membranes. Reducing SDS-PAGE shows a reduction from 460 kDa (left lane) to approximately 400 kDa (right lane) after treatment with peptide N-glycosidase F (PNGase F).

FIGS. 5A-B shows the extracellular modules of cubilin. FIG. 5A shows schematic representation of the 460 kDa receptor (designated cubilin) and related developmental control proteins, human bone morphogenic protein-1 (BMP-1), human tumor necrosis factor stimulating gene 6 (TSG-6), pig spermadhesin aqn3, and the Drosophila protein tolloid. The EGF repeats and CUB domains encode the whole protein except the 110 residues after the signal peptide. FIG. 5B shows a dot plot display of the high internal homology of the CUB domains in cubilin.

FIG. 6A shows alignment of the EGF repeats in cubilin and homologous repeats in CUB domain-containing proteins (Bmp-1, tolloid protein, C1s) and in human fibrillin-1 (SEQ ID Nos. 6-20). The consensus residues for calcium binding are indicated at the bottom of the figure. FIGS. 6B-6D show alignment of cubilin CUB domains and CUB domains in the development control proteins shown in FIG. 5, Xenopus laevis Uvs-2 and human C1s (SEQ ID Nos. 21-36).

FIG. 7 shows release of membrane-associated cubilin by non-enzymatic and non-solubilizing procedures. Western blot analysis with anti-cubilin antibody (top panel) and anti-megalin antibody (bottom panel). Lane 1: yolk sac BN cells; lane 2: ileal mucosa; lane 3: membrane phase after mechanical grinding of renal cortex; lane 4: fluid phase after mechanical grinding of renal cortex membranes, lanes 5-11: fluid phase of renal cortex membranes after a 1-h incubation in PBS (lane 5), PBS, phosphorylethanolamine, and heparin (lane 6), PBS, phosphorylethalolamine, heparine, and EDTA (lane 7), PBS and heparin (lane 8), PBS and EDTA (lane 9), PBS, heparin, and EDTA (lane 10), PBS and phosphorylethanolamine (lane 11).

FIG. 8 shows immunocytochemical localization of megalin (10 nm gold) and cubilin (5 nm gold) in the apical part of rat renal proximal tubule cell (FIG. 8A) and epithelial cell of rat yolk sac (FIG. 8B). The two proteins are colocalized in apical endocytic invaginations (AI), small (SE) and large (LE) endosomes, the small gold-particles (cubilin) being indicated by large arrowheads. Colocalization is also seen in dense apical tubules (cubilin, small arrowheads). Microvilli (MV) of the proximal tubule are labeled for both proteins, whereas very little labeling is seen on yolk sac microvilli. Bars, 0.1 μm.

FIG. 9 shows binding of cubilin to megalin as determined by affinity chromatography. ¹²⁵I-cubilin was applied to a megalin-Sepharose-4B column (•) or a blank Sepharose-4B column (o). The inset demonstrates autoradiography of SDS-PAGE of ¹²⁵I-cubilin and the eluted fraction 13 (#13). Bound radioactivity was eluted by the addition (arrow) of 10 mM EDTA to the running buffer.

FIGS. 10A-B shows characterization of the cubilin-megalin interaction by surface plasmon reasonance analysis. Rabbit megalin was immobilized to a sensor chip and the on rates and off rates for the binding of cubilin was recorded by flow of 20 nM purified cubilin along the chip surface. For control, cubilin was subjected to a blank chip. The values displayed are the recordings from the megalin-chip subtracted from the recordings from the blank chip. FIG. 10A shows sensorgram of the binding of cubilin to megalin. The binding curves in the presence of 10 mM EDTA or after prebinding of RAP to megalin are also shown. FIG. 10B demonstrates the formation of an IF-B₁₂-cubilin megalin complex by subsequent flow with cubilin, running buffer and IF-B₁₂. Evaluation of the binding data suggests a complex binding. By fitting the binding data to a one-binding-site model a K_(d) of 7 nM was measured.

FIGS. 11A-B shows multiple lines of evidence suggesting that cubilin binds light chains: FIG. 11A shows Western blot analysis with a holo-gp280 polyclonal antiserum (left gel) and SDS-PAGE result (right gel). Cubilin at 460 kDa is accompanied by a prominent band at the region of 56 kDa. FIG. 11B shows coomassie-stained two-dimensional gel with pH gradient from 4-8 on abscissa and molecular weight on the ordinate which demonstrates relative protein abundance.

FIGS. 12A-C shows direct binding analysis of cubilin and myeloma light chains by surface plasmon resonance. FIG. 12A shows binding of cubilin to immobilized κ-light chains is dose dependent with rapid low affinity association and dissociation kinetics. FIG. 12B shows competition experiment which further demonstrates the specificity of binding of cubilin to immobilized κ-light chains. A sample of cubilin (100 nM) was incubated with κ-light chains (10 or 490 μM), or κ-light chains (10 or 490 μM) prior to injecting the sample over the κ-light chain surface. The binding of cubilin to the immobilized surfaces was reduced in the presence of κ- or λ-light chains in a dose-response fashion. Data are representative of experiments with 4 light chains on 3 chips. FIG. 12C shows effect of temperature on binding of cubilin (110 nM) to λ-light chains surface. LC, light chains: RU, response units.

FIGS. 13A-B shows displacement of light chain binding to rat renal cortical brush-border membranes by anti-cubilin and megalin antisera. FIG. 13A shows that anti-cubilin polyclonal antiserum (•) inhibits ¹²⁵I-labeled λ-light chain binding to rat renal brush-border membrane vesicles, but megalin antiserum (o) had no effect. FIG. 13B shows vesicle-by-vesicle analysis of FITC-light chain binding by flow cytometry. Each panel depicts 2,000 vesicles, and each dot represents one vesicle. FITC-fluorescence on the abscissa is displayed against vesicle size on the ordinate. Representative of n=8. Note most but not all vesicles bind FITC-light chains in the left panel, and anti-cubilin antiserum reduces binding (right panel).

FIGS. 14A-B shows effects of anti-cubilin antibody on light chain endocytosis. FIG. 14A shows FITC-κ-light chain endocytosis by yolk sac epithelial cells (BN/MSV) at 30 min. FIG. 14B shows time course of the endocytosis of FITC-λ-light chain in BN/MSV cells in the presence and absence of anti-cubilin antibody over 40 min. LC, light chain; Ab, antibody.

FIGS. 15A-B shows direct effects of light chains on endosomal fusion reconstituted in vitro. The λ_light chains were loaded into rat renal cortical intermicrovillar cleft at 400 _M by addition to the homogenization buffer. Fusion reconstituted in vitro in light chain-loaded membranes was inhibited compared with albumin-loaded control membranes. Values are mean±standard error for n=8, p<0.05 by unpaired t-test.

FIG. 16 shows fluorescein-light chain binding to renal brush-border membrane vehicles in the presence of gentamicin by flow cytometry, demonstrating megalin is a light chain receptor.

FIGS. 17A-B shows extra renal expression of cubilin. FIG. 17A shows RT-PCR with 2 sets of primers. Lanes 1, 3, 5, 7 and 9 used a primer (tgcctaccacagcccaaatga, SEQ ID No. 37) located in one of the 3′ CUB domains; and lanes 2, 4, 6, 8 and 10 used another primer (agagccacaatgactgcag, SEQ ID No. 38) located in the end of the EGF regions. Lanes 1-2: RNA from spleen; lanes 3-4: RNA from brain; lanes 5-6: RNA from liver; lanes 7-8: RNA from heart; lanes 9-10: RNA from Brown Norway (BN) rat immortalized yolk sac epithelial cells; lane 11: RNA from immortalized opossum kidney (OK) cells with megalin primers (SEQ ID Nos. 39-40). FIG. 17B shows RT-PCR with cubilin primers (SEQ ID Nos. 37-38). Lane 1: kidney; lane 2: thyroid.

FIG. 18 shows urine cubilin. Normal rat urine (lane 1) and 2 separate preparations of rat renal brush border (lanes 2-3) were separated by PAGE-SDS, transferred onto nitrocellulose and probed with anti-cubilin antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses the molecular characterization of the 460 kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B₁₂ complexes in the intestine and kidney. The 3603 amino acid rat sequence has one cluster of 8 EGF-type domains followed by a cluster of 27 CUB domains accounting for 88% of the protein mass. The receptor, cubilin, has no similarity to known endocytic receptors. Instead, it displays homology to EGF and CUB domain-containing proteins involved in fetal development. Cubilin is a peripheral membrane protein which can be released from renal cortex membranes by non-enzymatic and non-solubilizing procedures. Electron microscopic immuno-gold labeling of rat yolk sac and renal proximal tubules revealed that the endocytic receptor megalin and cubilin strictly colocalize in the endocytic apparatus. Megalin-affinity chromatography and surface plasmon analysis demonstrated a calcium-dependent high affinity binding of cubilin to the extracellular part of megalin which thereby may assist the intracellular trafficking of this novel type of receptor.

Myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, however, the molecular identity of the receptor has not been characterized. The present studies provide several lines of evidence to identify cubilin (gp280), a giant glycoprotein receptor, which is preferentially expressed in endocytic scavenger pathways and which has potent effects on endosomal trafficking, as an endocytic receptor for immunoglobulin light chains. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. The data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.

Agents that inhibit binding of light chains are ligands of cubilin. More generally, the proteins present in the urine of patients or dogs deficient in cubilin contain a variety of proteins including albumin which constitute ligands.

Independent evidence also suggests that light chains are ligands for megalin. These studies are important, both to understand the complex interactions of toxic and physiological ligands on proximal tubule scavenger pathway receptors, as well as the eventual development of clinical protective agents for nephrotoxic damage mediated by ligands for cubilin and/or megalin.

If appearing herein, the following terms shall have the definitions set out below.

As used herein, the term “cDNA” shall refer to the DNA copy of the mRNA transcript of a gene.

As used herein, the term “derived amino acid sequence” shall mean the amino acid sequence determined by reading the triplet sequence of nucleotide bases in the cDNA.

As used herein the term “screening a library” shall refer to the process of using a labeled probe to check whether, under the appropriate conditions, there is a sequence complementary to the probe present in a particular DNA library. In addition, “screening a library” could be performed by PCR.

As used herein, the term “PCR” refers to the polymerase chain reaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, as well as improvements now known in the art.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription and Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984).

A “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

A “signal sequence” can be included near the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

The term “oligonucleotide”, as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence or hybridize therewith and thereby form the template for the synthesis of the extension product.

A cell has been “transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75% (preferably at least about 80%, and most preferably at least about 90% or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

A “heterologous” region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to untraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

Proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, 90Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized calorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

A particular assay system developed and utilized in the art is known as a receptor assay. In a receptor assay, the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantitiy of both the label after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.

An assay useful in the art is known as a “cis/trans” assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex. Thus, for example, if it is desired to evaluate a compound as a ligand for a particular receptor, one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted. If the compound under test is an agonist for the receptor, the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene. The resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands. The foregoing protocol is described in detail in U.S. Pat. No. 4,981,784.

As used herein, the term “host” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. A recombinant DNA molecule or gene which encodes cubilin protein of the present invention can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a vector containing coding sequences for the gene which encodes cubilin protein of the present invention for purposes of prokaryote transformation.

Prokaryotic hosts may include E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells.

In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

The invention includes a substantially pure DNA encoding a cubilin protein, a strand of which DNA will hybridize at high stringency to a probe containing a sequence of at least 15 consecutive nucleotides of SEQ ID NO:1. The protein encoded by the DNA of this invention may share at least 80% sequence identity (preferably 85%, more preferably 90%, and most preferably 95%) with the amino acids listed in SEQ ID NO. 2. More preferably, the DNA includes the coding sequence of the nucleotides of SEQ ID NO:1, or a degenerate variant of such a sequence.

The probe to which the DNA of the invention hybridizes preferably consists of a sequence of at least 20 consecutive nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 100 nucleotides or more (up to 100%) of the coding sequence of the nucleotides listed in SEQ ID NO. 1 or the complement thereof. Such a probe is useful for detecting expression of cubilin in a human cell by a method such as a method including the steps of (a) contacting mRNA obtained from the cell with the labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA.

This invention also includes a substantially pure DNA containing a sequence of at least 15 consecutive nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of the nucleotides listed in SEQ ID NO. 1.

By “high stringency” is meant DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65° C. at a salt concentration of approximately 0.1×SSC, or the functional equivalent thereof. For example, high stringency conditions may include hybridization at about 42° C. in the presence of about 50% formamide; a first wash at about 65° C. with about 2×SSC containing 1% SDS; followed by a second wash at about 65° C. with about 0.1×SSC.

By “substantially pure DNA” is meant DNA that is not part of a milieu in which the DNA naturally occurs, by virtue of separation (partial or total purification) of some or all of the molecules of that milieu, or by virtue of alteration of sequences that flank the claimed DNA. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein. Also included is a recombinant DNA which includes a portion of the nucleotides listed in SEQ ID NO. 1 encoding an alternative splice variant of cubilin.

The DNA may have at least about 70% sequence identity to the coding sequence of the nucleotides listed in SEQ ID NO:1, preferably at least 75% (e.g. at least 80%); and most preferably at least 90%. The identity between two sequences is a direct function of the number of matching or identical positions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then they are identical at that position. For example, if 7 positions in a sequence of 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. The length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).

The present invention comprises a vector comprising a DNA sequence coding for a human cubilin protein and said vector is capable of replication in a host which comprises, in operable linkage: a) an origin of replication; b) a promoter; and c) a DNA sequence coding for said protein. Preferably, the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID No. 1. A “vector” may be defined as a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid. Vectors may be used to amplify and/or express nucleic acid encoding cubilin protein. An expression vector is a replicable construct in which a nucleic acid sequence encoding a polypeptide is operably linked to suitable control sequences capable of effecting expression of the polypeptide in a cell. The need for such control sequences will vary depending upon the cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter and/or enhancer, suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Methods which are well known to those skilled in the art can be used to construct expression vectors containing appropriate transcriptional and translational control signals. See for example, the techniques described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press, N.Y. A gene and its transcription control sequences are defined as being “operably linked” if the transcription control sequences effectively control the transcription of the gene. Vectors of the invention include, but are not limited to, plasmid vectors and viral vectors. Preferred viral vectors of the invention are those derived from retroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpes viruses.

By a “substantially pure protein” is meant a protein which has been separated from at least some of those components which naturally accompany it. Typically, the protein is substantially pure when it is at least 60%, by weight, free from the proteins and other naturally-occurring organic molecules with which it is naturally associated in vivo. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure cubilin protein may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding an cubilin polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography such as immunoaffinity chromatography using an antibody specific for cubilin, polyacrylamide gel electrophoresis, or HPLC analysis. A protein is substantially free of naturally associated components when it is separated from at least some of those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be, by definition, substantially free from its naturally associated components. Accordingly, substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes, or any other organism in which they do not naturally occur.

In addition to substantially full-length proteins, the invention also includes fragments (e.g., antigenic fragments) of the cubilin protein (SEQ ID No. 2). As used herein, “fragment,” as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments of the cubilin protein can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant cubilin protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment of cubilin, or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of cubilin (e.g., binding to an antibody specific for cubilin, or binding to a known ligand of cubilin) can be assessed by methods described herein. Purified cubilin or antigenic fragments of cubilin can be used to generate new antibodies or to test existing antibodies (e.g., as positive controls in a diagnostic assay) by employing standard protocols known to those skilled in the art. Included in this invention are polyclonal antisera generated by using cubilin or a fragment of cubilin as the immunogen in, e.g., rabbits. Standard protocols for monoclonal and polyclonal antibody production known to those skilled in this art are employed. The monoclonal antibodies generated by this procedure can be screened for the ability to identify recombinant cubilin cDNA clones, and to distinguish them from known cDNA clones.

Further included in this invention are cubilin proteins or fragments which are encoded at least in part by portions of SEQ ID NO. 2, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of cubilin sequence has been deleted. The fragment, or the intact cubilin polypeptide, may be covalently linked to another polypeptide, e.g. which acts as a label, a ligand or a means to increase antigenicity. The lack of cross inhibition of a number of ligands suggests that specific therapeutic components can be produced.

Also within the invention is a method of detecting cubilin protein or fragment in a biological sample, which includes the steps of contacting the sample with the labelled antibody, e.g., radioactively tagged antibody specific for cubilin, and determining whether the antibody binds to a component of the sample.

A standard Northern blot assay can be used to ascertain the relative amounts of cubilin mRNA in a cell or tissue obtained from a patient, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art. This Northern assay uses a hybridization probe, e.g. radiolabelled cubilin cDNA, either containing the full-length, single stranded DNA having a sequence complementary to SEQ ID NO. 1, or a fragment of that DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 100 consecutive nucleotides in length). The DNA hybridization probe can be labelled by any of the many different methods known to those skilled in this art. RNA probes can also be similarly utilized.

The present invention is also directed to a vector capable of expressing the DNA of the present invention adapted for expression in a recombinant cell and regulatory elements necessary for expression of the DNA in the cell. Preferably, the vector contains DNA encoding a cubilin protein having the amino acid sequence shown in SEQ ID No. 2.

The present invention is also directed to a host cell transfected with the vector described herein, said vector expressing a cubilin protein. Representative host cells include consisting of bacterial cells, mammalian cells and insect cells.

The present invention is also directed to a isolated and purified cubilin protein or fragment coded for by DNA selected from the group consisting of: (a) isolated DNA which encodes a cubilin protein or fragment; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes a cubilin protein or fragment; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a cubilin protein or fragment. Preferably, the isolated and purified cubilin protein has the amino acid sequence shown in SEQ ID No. 2, and the fragment has amino acid sequence consisting of one or more of the sequences selected from the group consisting of SEQ ID Nos. 21-27.

The present invention is also directed to a method of detecting expression of the cubilin protein or fragment, comprising the steps of: (a) contacting mRNA obtained from a sample with a labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA.

The present invention is further directed to a pharmaceutical composition comprising the cubilin protein or fragment and a pharmaceutically acceptable carrier. Such composition can be used for treating or reducing nephrotoxicity or other types of toxicity in an in-need individual.

In an additional embodiment, the present invention is directed to a receptor for a variety of ligands, comprising a cluster of EGF repeats and a cluster of CUB domains. Specifically, the receptor is cubilin and ligand selected from the group consisting of immunoglobulin light chain, myoglobin, intrinsic factor-vitamin B₁₂, metallothionein, β-2-microglobulin, amyloid, hemoglobin, haptoglobin, interferon, insulin, cytochrome c, lysozyme, transferrin, transthyretin, polybasic drugs, low density lipoprotein, high density lipoprotein and receptor related protein. A representative example of a polybasic drug is gentamicin. Representative examples of immunoglobulin light chain include κ-light chain and λ-light chain.

In still yet another embodiment of the present invention, there is provided a method of detecting renal damage by measuring the level of cubilin in the urine of an individual suspected to have such damage. If the urinary cubilin level is lower than that of a normal individual, the test individual might have chronic renal damage, on the other hand, the test individual might have renal damage of acute origin if the urinary cubilin level is higher than that of a normal individual.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1 Animals, Reagents and Antibodies

Male Sprague Dawley rats (200-250 gm) were from Sasco, Omaha, NE, and all other reagents were from Sigma Chemical company (St. Louis Mo.) unless otherwise stated. Polyclonal antibodies were raised against proteins purified by immuno-affinity chromatography using reported monoclonal antibodies coupled to Sepharose 4B (Baricault et al., 1995; Sahali et al., 1988; Sahali et al., 1993). These antibodies are monospecific by immunoblotting on whole brush border preparations and by immuno precipitation of biosynthetically labeled yolk sac epithelial cells in culture (Sahali et al., 1993), and bind the cytosolic domains of the protein (Hammond et al., 1993). Control antisera included normal rabbit antiserum, and polyclonal rabbit antiserum to the neurokinin-1/substance P, NK1, receptor (from Professor Jean-Yves Courard, Gif.-Sur-Yvette, France).

EXAMPLE 2 Sequencing and Estimation of N-Linked Carbohydrate of Cubilin

CNBr fragments and tryptic digests of a 100 kDa CNBr fragment of purified rat cubilin were purified by reverse phase HPLC and seven isolated peptides subjected to Edmann degradation using an Applied Biosystems 477 A sequencer equipped with a 120 A on-line chromatograph. A cross-flow reaction and the Doublot reaction and conversion cycles were used. Deglycosylation with peptide N-glycosidase F (PGNase F) of 5 μg of purified rabbit intrinsic factor was carried out as described (Jensen et al. 1992).

EXAMPLE 3 cDNA Cloning, Sequencing and Northern Blotting

Total RNA was extracted from renal cortex and BN cells using Trizol (Gibco, Life Sciences) as described by the manufacturer. mRNA required for library construction was isolated using the Qiagen Oligotex kit. Northern blots were made with 1 μg of mRNA and revealed with ³²P labeled riboprobes (bp 1205 to 1645 and bp 1702 to 2175).

Four libraries were used. Two conventional libraries were constructed in the laboratory using cDNAs synthesized by oligo dT and random priming of polyA-selected RNA from yolk sac derived BN/MSV epithelial cells using the superscript Kit (Gibco Life Sciences). After ligation to EcoR1 adaptors and size fractionation, they were introduced in λZap or λgt11 EcoR1 site. Subsequently screening was performed on a commercial λZap cDNA library (Stratagene) prepared from yolk sac derived L2 epithelial cells. Finally to identify the 5′ end, a library was constructed in λgt11 using the 5′ Cap Finder library from Clontech. Immunoscreening was carried out on the λZap-BN library using previously reported polyclonal antibodies to gp280. cDNA probes were constructed from known sequences by PCR using a 1/19 mixture of digoxygenin labeled nucleotide (Boehringer) and used to identify overlapping clones.

RACE was carried out using Marathon ready cDNA prepared from rat renal cortex (Clontech). Specific primers were from bp 838-859 (SEQ ID NO. 3) for 5′ RACE and bp 6872-6891 (SEQ ID NO. 4) and bp 7152-7172 (SEQ ID NO. 5) for 3′ RACE. Inserts were prepared by the ex vivo excision system for λzap clones (Stratagene). cDNAs from λgt11 clones were isolated by EcoR1 digestion and inserted in Bluescript. Sequencing was carried out by cycle sequencing in both directions with IRD-41 labeled primers and the sequence reaction were analyzed on a LICOR 4000 automatic sequencer.

EXAMPLE 4 Release of Cubilin from Renal Cortex Membranes

Rat renal cortex (0.6 g) was suspended in 3 ml PBS, pH 7.4, containing 0.1 mM phenylmethylsulfonylfluoride and Pefablock (Boehringer) and homogenized on ice using an ultrathorax homogenizer (23,000 rpm/min) for 20 sec. The homogenate was centrifuged at 20,800×g for 20 min. The saline soluble and saline insoluble samples were analyzed by immunoblotting with anti-cubilin and anti-megalin monoclonal antibodies (Birn et al. 1997). The amounts loaded on the gels were adjusted so that both fractions were derived from 20 μg of original cortex. IF-B₁₂ affinity chromatography of the fluid phase was performed as described (Birn et al. 1997) except that the buffer contained no detergent.

Rabbit renal membranes were prepared as described (Moestrup et al. 1993). For release of cubilin, 2 mg of membranes were incubated in 525 μl of PBS, 250 units/ml heparin (LEO, Denmark), 20 mM EDTA or 5 mM phosphatidylethanolamine (Sigma) for 1 h at 22° C. followed by centrifugation at 20,800×g for 20 min.

EXAMPLE 5 Immunocytochemistry

Rat kidneys were fixed by retrograde perfusion through the abdominal aorta with 8% paraformaldehyde in 0.1 M sodium carcodylate buffer, pH 7.2. The tissue was trimmed into small blocks, further fixed by immersion for 1 hour in the same fixative, infiltrated with 2.3 M sucrose containing 2% paraformaldehyde for 30 minutes and frozen in liquid nitrogen. Rat embryos at day 12 of gestation were dissected free of the decidua and parietal layer to expose yolk sac epithelial cells. The tissue was then fixed by immersion and further processed as described above. For electron microscopy, 70 to 90 nm cryosections were obtained at −100° C. with an FCS Reichert Ultracut S cryoultramicrotome as described (Christensen et al. 1995). For double immunolabeling, the sections were incubated with the two primary antibodies overnight at 4° C. after preincubation in PBS containing 0.05 M glycine and 1% bovine serum albumin. Sheep anti-rat megalin serum (Moestrup et al. 1993) was diluted 1:200,000 and mouse monoclonal MAB75 (2 μg/ml) against cubilin (Sahali et al 1988). The sections were then incubated for 30 minutes with rabbit anti-sheep serum 1:20,000 (Dako A/S, Glostrup, Denmark), and finally incubated with 10 nm goat anti-rabbit gold particles and 5 nm goat anti-mouse gold particles (BioCell, Cardiff, UK). The sections were embedded in methylcellulose and studied in a Philips CM100 electron microscope. As controls, sections were incubated with secondary antibodies alone or with non-specific monoclonal antibodies or sheep antiserum.

EXAMPLE 6 Binding of ¹²⁵I-Cubilin to Megalin

Megalin was immobilized to CNBr-Sepharose 4B (Pharmacia, Uppsala, Sweden) at a density of 0.5 mg megalin/ml gel. Cubilin was iodinated (10⁶ Bq/μg) by the iodogen method (Pierce). The ¹²⁵I-labeled cubilin was purified by S-300 (Pharmacia) gel filtration and 10⁶ cpm was loaded on the megalin-column. After wash with binding buffer (20 mM Hepes, 150 mM NaCl, 2 mM CaCl₂, pH 7.8), bound radioactivity was eluted with the same buffer supplemented with 10 mM EDTA, counted and analyzed by SDS-PAGE.

EXAMPLE 7 Megalin-Cubilin Interaction Analysis by Surface Plasmon Resonance

Surface plasmon resonance measurements were performed on a BIAcore 2000 instrument (Pharmacia, Sweden). BIAcore sensor chips (type CM5, Pharmacia) were activated with 1:1 mixture of 0.2 M N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide and 0.05 M N-hydroxysuccimine in water. Rabbit megalin was immobilized as described (Moestrup et al., 1996) at a concentration of 40 μg/ml in 10 mM sodium acetate, pH 4.5 and the remaining binding sites were blocked with 1 M ethanolamine, pH 8.5. The flow buffer was 10 mM Hepes, 150 mM NaCl and 1.5 M CaCl₂, 1 mM EDTA, pH 7.4. The binding data were analyzed using the BIAevaluation program.

EXAMPLE 8 Preparation of Light Chains

Four species of light chains, two κ and two λ, were isolated and purified from the urine of four different patients with myeloma, as described (Batuman et al., 1990; Batuman et al., 1997). The purity and the immunologic identity of light chains were confirmed by SDS-PAGE and Western blotting. One of the λ-light chains and the κ-light chain used here were the same light chains used to demonstrate receptor-mediated endocytosis by radioisotope techniques. Competition experiments were initially conducted using radioiodinated λ-light chain, iodinated by the Iodobead method as reported (Batuman et al., 1990; Batuman et al., 1997). It was later switched to competition experiments with fluorescein isothiocyanate (FITC) conjugated κ-light chain. FITC conjugation was performed using FluoroTag FITC Conjugation Kit (Sigma ImmunoChemicals, St. Louis, Mo.).

EXAMPLE 9 Preparation of Renal Brush-Border Membrane Vesicles and Cortical Intermicrovillar Clefts

Rat renal cortical brush border membrane vesicles, inside/in, were isolated by magnesium precipitation technique as described (Batuman et al., 1990; Hammond et al., 1985). Rat renal cortical intermicrovillar clefts were prepared from kidneys harvested from anesthetized rats, utilizing differential Percoll gradient centrifugation and magnesium precipitation. It was also shown that the intermicrovillar clefts form vesicles “oriented inside out” in vitro during homogenization and can capture internally components added to the homogenization buffer.

EXAMPLE 10 Preparation of Cubilin

Intermicrovillar clefts prepared from renal cortices were biotinylated on the cytosolic facade using NHS-biotin (Winearls, 1995). Cubilin and the associated proteins were purified by immunoaffinity chromatography MAb 75 was coupled to CNBr-activated Sepharose 4B (Pharmacia, Saint Quentin en Yvelines, France) as previously described (Sahali et al., 1988; 1993). Protease inhibitors were added at all steps.

EXAMPLE 11 Competition Between Light Chains and Anti-Cubilin and Megalin Anti-Sera for Rat Renal Brush Border Membrane Binding

Binding of either [¹²⁵I]-labeled or FITC conjugated light chain was investigated in the presence of up to 100,000 fold serial dilutions of anti-cubilin antibodies (Baricault et al., 1995). Equal dilutions of bovine serum albumin served as controls. With the radio-labeled light chain, binding was assayed in a gamma counter as described (Batuman et al., 1990; 1997). Binding of FITC-conjugated light chain was assayed by flow cytometry using small particle techniques on a Becton-Dickinson FACStar flow cytometry with a Consort 30 computer and WinMidi software (Hammond et al., 1994; Sahali et al., 1988; 1993). The analog-to-digital conversion of fluorescence measurements on each particle passes through a logarithmic amplifier such that florescence is expressed on a log scale.

EXAMPLE 12 Surface Plasmon Resonance Analysis of Light Chain/Cubilin Interaction

κ- or λ-light chains were immobilized via free amine groups to the dextran matrix of CM5 sensor chips activated by a 1/1 mixture of NHS and EDC. Unreacted sites were blocked with 1M ethanolamine, pH 8.5, (Jonsson et al., 1991; Sanders et al., 1988). The immobilization was conducted at 25° C. using 10 mM HEPES, 2 mM CaCl₂, 150 mM NaCl, 0.005% NP-40, pH 7.4 as the flow buffer. Then 10 mM acetate pH 4.8 was used for electrostatic pre-concentration of the protein. Different densities of κ- or λ-light chains were immobilized to three of the four flow cells; the remaining flow cell was activated and blocked with no light chains immobilized for use as a control surface. Binding experiments were carried out using a BIACORE 2000 instrument.

EXAMPLE 13 Identification by Two-dimensional Gel Electrophoresis and Microsequencing of Proteins Associated with Immunopurified Cubilin

The approach used involved three steps: 1) biotinylation of intermicrovillar membranes, 2) immunoisolation of cubilin, and 3) identification of bound proteins by microsequencing. Two-dimensional electrophoresis w as performed according to the method of O'Farrell (O'Farrell, 1975) by Kendrick Labs, Inc. (Madison, Wis.). Proteins other than cubilin observed on two-dimensional gels prepared from the eluate of detergent solubilization of intermicrovillar clefts were identified by microsequencing. For this purpose, three gels were run in parallel, and stained with Coomassie D. The two most abundant spots at MW 56 and 24 kDa from each gel were cut out and the material pooled. The peptides derived from the eluted proteins by C-leu digestion were separated by HPLC, and internal peptides sequenced (Ferrara et al., 1993).

EXAMPLE 14 Effect of Light Chains on Endosomal Fusion

To determine if light chains had a direct effect on membrane fusion, rat renal cortical intermicrovillar clefts were prepared as described (Hammond et al., 1997; 1994) and loaded with 400 mM light chains by addition of the light chains to the homogenization buffer. Fusion of these light chain loaded membranes was compared to control membranes loaded with the same concentration of albumin. All fluorescence measurements were corrected per mg of protein, and fusion reconstituted in vitro in cuvettes (Hammond et al., 1994). Data are expressed as mean +standard error of the mean throughout the application. Statistical analysis was performed by analysis of variance and Bonferroni or Scheffe's post hoc comparison.

EXAMPLE 15 Culture of Rat Visceral Yolk Sac Cells and Internalization Experiments

The yolk sac epithelial cell line (BN/MSV) was derived from yolk sac teratocarcinoma induced by fetectomy and placental injection of mouse sarcoma virus (Sahali et al., 1988). When grown under conventional conditions in modified Eagle's medium, supplemented with 2.5 mM L-glutamine, 10% fetal calf serum, and an antibiotic cocktail (penicillin, streptomycin, and Fungizone), the cells form a domed monolayer and express abundant cubilin (Sahali et al., 1988).

EXAMPLE 16 Effect of Anti-Cubilin Antiserum on Endocytosis of Light Chains

Internalization experiments were conducted by exposing confluent yolk sac cells in 24-cell plates to 50 μm FITC-conjugated light chain. These cells were selected for endocytosis experiments because cubilin expression is 100-fold greater than cultured proximal tubule cells (Sahali et al., 1988). Cells were allowed to endocytose FITC-light chain at various intervals for up to 40 minutes at 37° C. with and without polyclonal anticubilin antibody at 1:1,000 dilution (added at time 0). This concentration is selected because it is 10-fold higher than the half-maximal inhibitory concentration of the antibody determined from the brush border binding inhibition experiments. Endocytosis is stopped by washing twice with PBS and removing light chain from medium. Cells are then trypsininzed, fixed in 1% formaldehyde, and suspended in PBS, and FITC incorporated into each cell is read in a Becton-Dickinson flow cytometer as described previously. Endocytosis curves are generated by plotting fluorescence units corrected for background against time. Excess unlabeled light chain was used to test for specificity, and bovine serum albumin was used as nonspecific protein control.

EXAMPLE 17 cDNA Cloning of Cubilin

By immunoscreening of the λ-Zap cDNA library from rat yolk sac BN cells (Le Panse et al. 1995), an initial 0.7 kb clone encoding a portion of cubilin was identified. The 5′ sequence of this clone was used to design two nested primers to perform 5′ RACE on kidney cDNA allowing identification of the 5′ end of cubilin. Using PCR-generated probes for further screening of yolk sac libraries a number of clones were identified. FIG. 1 schematizes three overlapping clones completely sequenced and used to construct the final cDNA. The last clone contained a polyadenylation signal and a poly A tail. The 3′ and 5′ ends of the 11.8 kb sequence were further confirmed respectively by sequencing a 3′ end RACE product and a λgtll clone selected from a Cap Finder library. Northern blot analysis of yolk sac mRNA (FIG. 2) identified a mRNA of the size as the cDNA.

EXAMPLE 18 Primary Structure of Cubilin

The assembled cDNA (SEQ ID no. 1) revealed an uninterrupted open reading. frame of 10.8 kb encoding a 20 amino acid signal peptide (in italic type) and a 3603 amino acid protein (SEQ ID NO. 2) with 42 potential N-glycosylation sites (FIGS. 3A-3C). The molecular size of the protein backbone was calculated to 397 kDa. The seven amino acid sequences determined by N-terminal microsequencing of tryptic and CNBr peptides were all identified in the translated sequence (bolded letters in FIGS. 3A-3C). The size of the protein was confirmed by SDS-PAGE (FIG. 4). Deglycosylation of the receptor by PNGase F increased its electrophoretic mobility corresponding to a size of 400 kDa. Compared to the 460 kDa size of the untreated protein this indicates a carbohydrate content of ˜13% of the receptor mass. FIG. 5A shows the predicted domain organization of the receptor. A stretch of approximately 110 amino acids with no apparent homology to known proteins is followed by a cluster of 8 EGF type B repeats which precedes 27 contiguous CUB domains accounting for 88% of the protein mass. The high degree of internal homology (overall similarity of 45%) between the CUB domains is evident from the dot plot display shown in FIG. 5B. A total of 76 disulfide bridges is predicted if all the extracellular modules fold normally. The only cysteine outside the CUB domains and EGF repeats, is located in the 110 amino acid N-terminal sequence. This cysteine might account for the partial, disulfide bond dependent dimerization of a minor part of purified receptor (Le Panse et al., 1995; Birn et al. 1997).

FIGS. 6A-6D show alignment of the EGF repeats and CUB domains of some of the most homologous regions of other proteins. Two of the EGF repeats (Nos. 2 and 4) contain the consensus sequence for Ca²⁺ binding and β-hydroxylation of Asp/Asn (Selander-Sunnerhagen et al. 1992). The 110 amino acids CUB domains contain 4 cysteines except for CUB domain 13 which is missing the first two cysteins suggested to form the upstream disulfide bond (Bork and Beckmann 1993). The high homology of the CUB domains of bone morphogenic factor, the Drosophila dorsal-ventral patterning gene product tolloid, the embryonic protein Uvs2 in Xenopus Laevis, tumor necrosis factor stimulating gene 6 (Tsg6), C1r/C1s and spermadhesin is seen in the two lower panels (FIGS. 6A-6D).

Save for the leader peptide, no sequence compatible with a transmembrane domain could be identified. This excludes the protein as a type 1 membrane protein or a glycosyl-phosphatidylinositol-anchored protein, which is synthesized with a cleavable hydrophobic C-terminal. Furthermore, since almost the entire protein sequence consists of extracellular modules it is very unlikely that the protein is a type II or III protein with a non-cleaved hydrophobic signal peptide inserted in the membrane (Levy 1996).

EXAMPLE 19 Cubilin is a Peripheral Membrane Protein

In order to verify that cubilin is a peripheral membrane protein, as predicted by the lack of a transmembrane segment and cytoplasmic tail, its release from renal cortex membranes by procedures which do not involve solubilization of the membranes or enzymatic treatment was investigated. FIG. 7 shows the identical size of the renal receptor and the receptor in yolk sac and intestinal mucosa (lanes 1-3). As seen in lane 4 vs. lane 3, approximately 50% of cubilin was released into the fluid phase by mechanical grinding of renal cortex in PBS, whereas megalin, the 600 kDa transmembrane protein expressed in the same tissues (Saito et al. 1994), was released in minimal amounts. Cubilin, which remained membrane-associated, was tightly bound but could be released partly by EDTA, heparin and, to a low extent phosphorylethanolamine (FIG. 7, lanes 5-11). Heparin and phosphorylethanolamine have been reported to bind to the spermadhesin CUB domains (Calvete et al. 1996; Dostolova et al., 1995). The same treatments released virtually no megalin (FIG. 7). The size of the released cubilin, as estimated by SDS-PAGE, was not different from the membrane associated cubilin.

EXAMPLE 20 Cubilin Traffics with and Binds Megalin

Previous studies have demonstrated megalin and cubilin in endocytic vesicles of the same absorptive epithelia in the intestine, kidney and yolk sac. FIG. 8 shows electron microscopic examination of rat yolk sac and kidney section subjected to double gold-labeling of megalin and cubilin using a sheep anti-megalin polyclonal antibody and a mouse anti-cubilin monoclonal antibody. The large gold particles label megalin antibody and the small particles label cubilin antibody. An almost identical localization of the two sizes of gold particles was seen. Formation of cubilin/megalin complexes was tested next.

FIG. 9 shows ¹²⁵I cubilin binds megalin covalently linked to Sepharose 4B. Bound radiolabel was released from the column by EDTA. Surface plasmon analysis (FIG. 10A) confirmed this binding. No difference in the dissociation of cubilin to megalin was seen in the pH interval 4-8. Binding of cubilin to megalin was reduced (75%) when RAP was prebound to megalin indicating that cubilin binds to the extracellular domain of megalin. Megalin-bound cubilin was still capable of binding IF-B₁₂ as shown by subjecting the megalin-chip to flow with IF-B₁₂ after the binding of cubilin (FIG. 10B). Thus, the response after adding IF-B₁₂ represents the formation of a megalin-cubilin-IF-B₁₂ complex. Control experiments showed no binding of IF-B,₂ to megalin.

EXAMPLE 21 Light Chains are Ligands for Cubilin

To identify candidate ligands with which cubilin interacts, a detergent extract of rat renal apical intermicrovillar clefts biotinylated on their cytosolic facade to affinity chromatography was subjected. The extract was passed through an immunoaffinity raised against the whole molecule. Western blot analysis of the eluate using the same antibody showed a single band at the region of 460-540 kDa, consistent with cubilin (FIG. 11A, left lane). Coomassie staining of a parallel gel revealed several additional bands (FIG. 11A, right lane). For further characterization, the proteins eluted from the column were separated by two-dimensional gel electrophoresis and transferred, and the spots were cut of the gels (FIG. 11B). Pooled material representing the same spot from multiple gels was C-leu digested, fragments separated by HPLC and microsequenced (Ferrara, et al., 1993). Proteins eluted from the column included cubilin (FIG. 11A, left at top of gel), a 56-kDa protein identified as the β-subunit of the H⁺-ATPase by the sequence VVDLLAPYA (FIG. 11B, #1), a 24-kDa protein identified as κ-light chains by the sequence (I/S)PQLLVYNA (FIG. 11B, #2), and an internal tropomycin control protein added exogenously to the gel (FIG. 11B, solid arrow). The 56 kDa protein was biotinylated suggesting cytosolic residence, and hence was not pursued as a ligand. The 24-kDa protein was not biotinylated, suggesting exofacial residence (Table 1).

TABLE 1 Analysis of Anti-gp280 Affinity Column Eluate Protein Commassie Anti-gp280 Cytosolic W.M. (kDa) Stain Western Biotinylation 540 + + +  56 + − +  24 + − −

Whether light chains are a ligand for cubilin, or were merely eluting from the antibody on the column remained uncertain. Analysis of cubilin binding to κ and λ-light chains using surface plasma resonance techniques provides direct evidence that cubilin binds light chains. A stock solution of cubilin was diluted serially with flow buffer and passed over the immobilized _-light chain surfaces for 5 minutes (50 μl at 10 μl/min., 25° C.), followed by monitoring the dissociation phase induced by introduction of cubilin free-flow buffer for 4 minutes (FIG. 12A). After 4 minutes, the cubilin bound to the surface had dissociated completely, so it was not necessary to regenerate the surface prior to the next injection. The sensorgrams were corrected for bulk refractive index changes by subtracting the response on the blank flowcell from the other flowcells. Cubilin bound to κ-light chains in a dose-dependent fashion (FIG. 12A).

To further demonstrate the binding specificity of the cubilin to the immobilized κ-light chains, a competition experiment was conducted. A sample of cubilin (100 nM) was incubated with κ-light chains (10 or 490 μM), or λ-light chains (10 μM) prior to injecting the sample over the κ-light chain surface. The binding of cubilin to the immobilized surfaces was reduced in the presence of κ-light chains in a dose-response fashion (FIG. 12B). Inhibition of cubilin binding to immobilized κ-light chains with 10 μM λ-light chains suggests κ and λ light chains share a common binding site on cubilin. This series of experiments was repeated with immobilized_λ-light chains, and four different light chains competing (two λ and two κ) with similar results (data not shown). These studies showed that cubilin bound λ-light chains in a dose-dependent fashion, and that binding was interfered with in a dose-response fashion by both free λ- and κ-light chains. In these studies, bovine serum albumin neither competed with light chains, nor bound to cubilin.

Binding of cubilin to κ-light chains was much greater at 37° C. than 25° C. (FIG. 12C), consistent with known thermal behavior of receptor-ligand interactions (Batuman et al., 1990). Hence, BIACORE surface plasmon resonance analysis allows for direct real time assay of the binding of myeloma light chains to cubilin, providing direct evidence that cubilin is a renal light chain receptor.

To determine whether light chains bind to cubilin present in brush-border membranes in its native membrane-bound form, antibody interference with light chain binding to rat kidney brush-border membrane vesicles, which are known to express cubilin (Sahali et al., 1988), was tested. Binding of [¹²⁵I]-labeled human λ-light chain to rat renal brush-border membrane vehicles is displaced by polyclonal antibodies to cubilin. The half-maximal inhibitory concentration of anti-cubilin antibody was observed at approximately 10,000 dilution (FIG. 13A, solid circles). In contrast, antiserum to megalin, which is known to bind these membranes (Moestrup et al., 1995), had no effect on the binding of this light chain (FIG. 13A, open circles), suggesting that this λ-light chain binds exclusively to cubilin.

At the maximal inhibitory concentration, the anti-cubilin antiserum displacement of λ-light chain approached 90%, confirming near exclusive binding of this light chain to cubilin. It was also observed that binding of human FITC-conjugated κ-light chain to rat renal brush-border membrane vesicles was displaced by polyclonal antibodies to cubilin as assayed by flow cytometry (FIG. 13B). Light chain binding (45.5±4.3 arbitrary fluorescent units, n=8) increased compared to unstained membranes (5.1±1.2 units, n=8, p<0.05), and was displaced by anti-cubilin (30.2±_(—)1.0 units, n=8, p<0.05). There was no effect on light chain binding by normal rabbit serum (42.9±1.7 units, n=8), or antiserum to the neurokinin-1/substance-P receptor (40.0±1.2 units, n=4), an irrelevant antibody which binds these membranes. This provides additional evidence that the competitive effect of cubilin antiserum on the binding of light chain is specific. Flow cytometry histograms of light chain binding on a vesicle-by-vesicle basis illustrate the effects of cubilin antisera on rat renal brush border binding of FITC-κ-light chains. Each histogram (FIG. 13B) displays 2000 vesicles as individual dots, with FITC fluorescence plotted against vesicle size. FITC-light chains bind most but not all brush borders (FIG. 13B, left panel). Cubilin antiserum displaced FITC light chain binding (FIG. 13B, right panel).

To examine the role of cubilin in light chain endocytosis, yolk sac cells were allowed to endocytose FITC-light chain in the absence and presence of anti-cubilin antiserum. These endocytosis experiments revealed a significant inhibitory effect but not total elimination of endocytosis (FIG. 14). Excess unlabeled light chain and anti-cubilin antibody reduced FITC-light chain endocytosis significantly (n=4, p<0.002, Mann-Whitney-U test), whereas albumin had no effect (FIG. 14A). Furthermore, a time course study showed that anti-cubilin antiserum inhibited light chain endocytosis significantly at all time intervals studied (FIG. 14B, n=3 each time period, p<0.0001). This time course experiment also showed that anti-cubilin antiserum eliminated the saturable pattern of endocytosis with apparent linearization of the uptake curve (FIG. 14B). This observation further supports that cubilin mediates light chain endocytosis in yolk sac cells. Less than complete inhibition of light chain endocytosis in the presence of anti-cubilin antiserum also indicates that, when this pathway is blocked, some light chain endocytosis occurs through alternate pathways, and that the cubilin-facilitated path is not exclusive endocytic pathway for light chains.

EXAMPLE 22 Function of Light Chains on Endosomal Fusion

To test whether myeloma light chains are functionally important in membrane trafficking and fusion events, intermicrovillar clefts were loaded with light chains by adding it to the homogenization buffer (Hammond et al., 1994). Fusion reconstituted in vitro in cuvettes was assayed by energy transfer, and results were normalized per milligram protein (Hammond et al., 1994; Jo et al., 1995). Fusion was significantly inhibited in membranes treated directly with light chains (111±89 arbitrary fluorescence units/mg protein, n=8) compared with albumin entrapped controls (1584±314, n=8, p<0.0003 by unpaired t-test, FIG. 15).

EXAMPLE 23 Myeloma Light Chains Bind Megalin

Given the abundance of megalin on the renal brush border membrane, if megalin is a light chain receptor this would predict that small polybasic drugs, such as gentamicin, which are known ligands for megalin, should compete for light chain binding to renal brush border membrane vesicles. To test this, rat renal brush border membrane vesicles were incubated in fluorescein-conjugated light chains with various concentrations of gentamicin. After washing, fluorescein-light chain binding to the membranes was analyzed by flow cytometry (FIG. 16). Estimate of half maximal binding concentration of gentamicin between 60 and 70 μM was placed in the middle of the curve. Gentamicin competes with fluorescent light chain binding to renal brush border membranes in a dose-dependent manner (control FITC-light chain binding 39.6±4.2 arbitrary fluorescence units, 10 μM gentamicin 45.1±3.1, 100 μM gentamicin 8.9±33.8*, 1000 μM gentamicin 6.3±1.9*, n=4, *p<0.05). This demonstrates that gentamicin competes with light chains for brush border membrane binding.

EXAMPLE 24 Extra Renal Expressipon of Cubilin

Cubilin was demonstrated to be expressed in extra-renal tissues as well (FIGS. 17A and 17B). Spleen, brain, liver, heart, kidney and thyroid are the possible sites where cubilin is expressed. Administering cubilin in a pharmaceutically acceptable carrier might lead to the reduction of toxicity and therefore protecting those sites.

EXAMPLE 25 Urine Cubilin

Cubilin was also detected in the urine (FIG. 18), which indicates that cubilin is released into the urine and the assay of urinary cubilin might be an excellent marker for detecting renal damage. Several conditions can be considered: 1) renal damage of acute origin may increase the excretion of a tubular protein such as cubilin and constitute a more sensitive and specific marker than adenosine deaminase (Iglesias et al., 1994; Parvez et al., 1990; Tolkoff-Rubin et al., 1987); 2) chronic renal damage with tubular atrophy may be associated on the other hand with a reduced expression of cubilin, the assessment/follow up of which may be useful to monitor the evolution of renal fibrosis; 3) because Imerslund Grasbeck (IG) patients do not all have proteinuria, it is likely that the syndrome is associated with various mutations of cubilin (for comparison one or two hundred have been described in familial hypercholesterolemia) which may be associated with variable levels of cubilin excretion. In fact the assay of IF-B₁₂ binding activity of the urine of IG patients has been studied (Dugue et al., 1998; Gueant et al., 1995); and 4) some diseases characterized by proteinuria of unknown cause may be due to a defect in cubilin, which may be absent and therefore not detectable in the urine, or excreted in large amounts if it is not gathered adequately to the membrane.

More generally it can be presumed that mutations of key receptors of trophoblastic cells, such as cubilin may account for a variety of pathologies. For instance, the ulk of fetal malformations, not accounted for by the known or suspected hereditary abnormalities, might be related to cubilin defect. Similarly, most cases of poor fetal development or fetal loss, which are of unknown origin up to date, might be caused by cubilin mutations.

The present study provides novel molecular information on cubilin, previously known as the yolk sac target antigen of teratogenic antibodies and the intestinal receptor for IF-B₁₂. The primary structure predicts 35 extracellular modules uniquely organized in a cluster of 8 EGF repeats followed by, from a molecular point of view, an huge cluster of 27 CUB domains which account for 88% of the mass of the protein. Northern and western blotting of kidney, yolk sac and intestine indicate no difference in size of the receptor in these organs.

The EGF type B repeats are similar to the carboxyl-terminal extracellular modules of megalin and low density lipoprotein receptor-related protein. Cubilin has otherwise very little homology to these two giant receptors, which also bind RAP and mediate endocytosis of a variety of ligands. Also cubilin does not display homology to sortilin, the 95 kDa putative vesicular sorting receptor, which also binds RAP (Petersen et al., 1997). The CUB domains conform to the description of Bork and Beckmann (1993) based on the analysis of 31 copies of a module initially identified in C1r and C1s components of complement and subsequently in a variety of proteins associated with fetal development. They consist of 110 amino acids defining a characteristic hydrophobicity pattern predicted to form antiparallel beta barrels (Dias et al. 1997). The four conserved cysteines, generally thought to form two S-S bridges (1-2, 3-4), are found in all but domain 13 of cubilin which lacks the first 2 cysteines as already described in the first CUB domains of C1r/s and the homologues MASP1/2. When analyzed individually, the CUB domains of cubilin are more closely related to those seen in developmental control proteins.

On the functional level there is compelling evidence that the CUB domains are involved in the binding of proteins, as described for the Ca²⁺-dependent formation of the C1 complex (Bosby and Ingham 1990), as well as for binding of phospholipids and carbohydrates, as demonstrated for spermadhesins (Calvete et al. 1996a and 1996b, Dostalova et al. 1995). In addition to the CUB domains, the EGF repeats might also account for some of the binding properties of cubilin. EGF repeats are widely expressed and involved in a number of receptor-ligand interactions (Davis 1990). Two of the EGF repeats in cubilin have the consensus sequence for calcium binding (Rao et al. 1995) and may be involved in the calcium-dependent binding of e.g. RAP or IF-B₁₂.

The lack of a transmembrane segment was surprising since in previous studies isolation of cubilin relied on the use of detergent solubilized membranes. Furthermore the previous studies showed that cubilin was internalized through clathrin coated organelles and recycled via dense apical tubules (Le Panse et al. 1995). This first suggested to reassess membrane tethering of cubilin. Early results was thus confirmed indicating that an intrinsic factor-B₁₂ binding protein (Cotter and Rothenberg 1976) and the target of teratogenic antibodies (Leung 1982) could be released at least in part from intestinal or renal tissue using mechanical dissociation in the absence of detergents. It was further showed that whereas membrane association was stable between pH 4 and 8 cubilin could be released by heparin, phosphorylethanolamine and EDTA. These observations, which indicate nonionic interactions with sugars and phospholipids, are in line with the membrane binding properties of spermadhesins which consist of a single CUB domain, lack a transmembrane segment, but are yet tightly bound to the surface of sperm cells via phospholipids (Dostalova et al. 1995). Another region of the same CUB domain binds to carbohydrates of zona pellucida, the extracellular investment surrounding the mammalian egg. The lectin binding characteristics of the spermadhesins are not fully characterized but include heparin and a variety of carbohydrates including Gal beta (1-4)-GlcNac and Gal beta (1-3)-GlcNac (Calvete et al. 1996,1997). In view of the 27 CUB domains present in cubilin this receptor may have multiple membrane attachments which may account for the inability to release all the membrane associated cubilin.

The identification of the components which link cubilin to the membrane is also essential for understanding its internalization and recycling. The present study suggested that the binding of cubilin to megalin is crucial for this process. Co-internalization of a receptor which lacks internalization signal(s) by means of another receptor has previously been shown. The GPI linked urokinase receptor can thus be endocytosed by coupling of urokinase receptor-bound urokinase/inhibitor complex to the LDL receptor-related protein (Nykjær et al. 1997, Conese et al. 1996). It is likely that a similar process can be mediated by megalin which can also bind the urokinase-inhibitor complex (Moestrup et al. 1993). Based on the strict colocalization of cubilin and megalin at the subcellular level and on the ability of megalin to bind cubilin in vitro, it was proposed that megalin is crucial for the internalization of cubilin and cubilin ligand complexes. After internalization, the ligand IF-B₁₂ is segregated from the receptor and directed to lysosomes for degradation of IF (Dan and Cutler 1994, Birn et al. 1997) whereas cubilin is recycled to the membrane. Since the cubilin/megalin complex is stable at pH 5, the two receptors might remain in complex during the entire recycling pathway at variance from the urokinase receptor which recycles to the plasma membrane without being linked to the LDL receptor-related protein (Nykjær et al. 1997).

Upon analysis of the effect of polyclonal megalin antibodies and RAP on the endocytosis of ¹²⁵I-IF-B₁₂ in cultured yolk sac cell, only a 10 to 15% reduction was found in uptake. This modest effect might be accounted for a short cell surface expression of megalin and cubilin due to rapid recycling of the two proteins and thereby a too short time for the cubilin-megalin to dissociate, a prerequisite for RAP to block binding. Furthermore, a continuous incubation with RAP will probably have no effect on intracellular receptors, since externally receptor-bound RAP is transported to lysosomes for degradation (Iadonato et al., 1993).

In order to further characterize the partnership of these two giant receptors, studies have been initiated to investigate cubilin trafficking in megalin deficient or megalin-mutated cells expressing cubilin. However, such analysis might be complex since recent data on megalin deficient mice indicate a key role of megalin for normal development of the endocytic apparatus in the proximal kidney tubules and for survival of the mice in general (Willnow et al. 1996).

The observation that the target of teratogenic antibodies contains CUB domains is of particular interest in view of the fact that these domains are often observed in developmentally regulated proteins. The mode of action of the teratogenic antibodies is not known but have been shown to inhibit endocytosis, thus reducing the amount of maternal proteins internalized and consequently the amount of protein derived amino acid which can be incorporated into embryonic tissue (Beckman et al., 1997; Lloyd, 1990, Le Panse et al., 1994). However, there is no direct evidence that a decreased amino acid supply is responsible for foetal malformations. Alternatively, the teratogenic effect might relate to a more specific disturbance of the materno-foetal barrier such as an impaired transfer of B₁₂ or of other nutrients. Interestingly, the pattern of antibody induced fetal malformations which includes abnormal cranio-facial development, in particular of the eyes and hypophysis (Sahali et al., 1988) resembles to some extent the holoprosencephalic syndrome induced by anti-cholesterol agents (Llirbat et al. 1997), knock out of the cholesterol-depedent Sonic hedgehog (shh) gene (Porter et al., 1996) or of the megalin gene (Willnow et al., 1996). It has been proposed (Herz et al., 1997) that the defective development of the central nervous system in megalin-deficient mice was related to a decreased megalin-mediated uptake of cholesterol-containing lipoproteins which in turn altered the addition of cholesterol to the shh protein. It is therefore possible that anti-cubilin antibodies could interfere with cholesterol uptake either directly or indirectly via binding of cubilin to megalin in the yolk sac.

The present study establishes cubilin as a novel type of peripheral membrane receptor with multiple potential sites for interaction with other proteins and membrane components. Cubilin can bind IF-B₁₂, RAP, megalin, and most likely calcium, phospholipids and carbohydrates (Table 2). However a number of ligands may remain to be identified in order to explain the role of the receptor in kidney function and its importance in fetal development.

TABLE 2 Ligands for Cubilin competition BN surface antibody with cell plasmon inter- known Ligand uptake resonance ference ligands light chains* X X X X myoglobin X X X metallothionein X X X haptoglobin X X polybasic drugs X X X Intrinsic factor X X X vitamin B₁₂ LDL X X HDL X X transferrin X RAP X X X albumin X X X X *as light chains compete with renal brush border membrane binding with multiple other medically and physiologically important proteins, this suggests that these proteins are also ligands for cubilin. This group of proteins include β2 microglobulin, amyloid, insulin, cytochrome c and interferon. LDL: Low density lipoprotein; HDL: High density lipoprotein; RAP: Receptor related protein.

Solution structure of spermadhesin PSP-I/PSP (Romero et al. Nat. Struct Biol 1997 10:78-788), a dimer which consists exclusively of two single CUB domains has recently been obtained. It reveals that CUB domains are characterized by 2 layers of 5 beta sheets, the top layer of one of the CUB domains contacting the lower layer of the other CUB domain in a manner that leaves preferentially exposed the less conserved beta turns which carry the ligand binding sites. If such an arrangement prevails in cubilin, it is likely to account for and one would predict a wide variety of ligands binding to distinct CUB domains.

Indeed the fact that RAP binds cubilin but does not inhibit binding of IF-B₁₂ complexes indicates that these 2 ligands bind to different sites and probably to different CUB domains. Similarly haptoglobin and light chains both bind cubulin but do not compete for binding. The modular structure of cubilin thus strongly suggests that it may be feasible to produce fragments of small size corresponding to one or a few CUB domains which can be used therapeutically: this type of fragments will bind selected ligands but preserve many/most other functions of cubilin.

The present studies also showed that cubilin, a giant receptor which participates in the endocytic scavenger pathway of the renal proximal tubule cells, binds and facilitates endocytosis of immunoglobulin light chains isolated from the urine of myeloma patients. Evidence that cubilin is a light chain receptor came from the analysis of eluates from an affinity column prepared with anti-cubilin antiserum in which cubilin coeluted with κ-light chain. The κ-light chain was definitively identified by microsequencing after isolation by two-dimensional electrophoresis. Several additional lines of evidence add weight to the hypothesis that cubilin is a light chain receptor. Competition experiments by anti-cubilin antiserum and surface plasmon resonance experiments both showed that all tested light chains bind to cubilin.

Surface plasmon resonance technology allowed direct analysis of the binding of light chains to cubilin. Several characteristics of the observed sensorgrams suggest that light chains bind cubilin specifically. First, cubilin bound to light chains in a temperature- and dose-dependent manner whether κ or λ-light chain is immobilized. Second, four species of non-immobilized light chains all interfered with binding in a dose-dependent manner. Third, the kinetics of binding and displacement were very similar to values reported using radioactive membrane binding techniques (Batuman et al., 1997; Driesbach et al, 1994; Marchalonis et al., 1992). Lastly, λ-light chains interfere with λ-light chain binding to cubilin and vice versa. This data revalidates the use of surface plasmon resonance technology to quantitate low affinity binding (Jonsson et al., 1991; Sanders et al., 1988).

As κ-light chains are 100-fold more abundant than λ-light chains in healthy animals and humans (Riedel et al., 1991), it is not surprising to observe κ-light chains eluting from the cubilin affinity column but not λ-light chains. The current surface plasmon resonance data provides direct evidence confirming and extending the observation made by membrane binding of light chains: both κ- and λ-light chains are ligands for cubilin.

Studies of classic binding kinetics utilizing Scatchard analysis demonstrate several ligands competing with light chain for brush border membrane binding. These ligands include lysozyme, insulin, cytochrome c, myoglobin and β₂-microglobulin (Batuman et al., 1990; 1997; Driesbach et al., 1994). Competition by low molecular weight proteins raise the probability that cubilin is a multi-ligand receptor responsible for the endocytosis and cellular trafficking of a number of proteins normally filtered in the glomerulus and catabolized in the kidney, extending the role of this scavenger pathway receptor to such diverse phenomena as rhabdomyolysis and insulin metabolism. The multiple putative ligands for cubilin reflect the precedent set by other giant glycoprotein receptors such as the low-density-lipoprotein receptor, megalin, and the α₂-macroglobulin receptor, which bind many ligands with a spectrum of affinities at multiple binding sites (Moestrup et al., 1994). Cloning data that reveal multiple EGF repeats and CUB domains strengthens this expectation.

Receptor kinetic studies have demonstrated that light chain binding to receptors in cultured proximal tubule cells is followed by endocytosis and ultimate lysosomal degradation. The present observations suggest that cubilin is a receptor that can mediate endocytosis of light chains in renal proximal tubular cells. Nearly 90% of the λ-light chain binding was displaced by anti-cubilin antibody. In contrast, anti-megalin antibody did not compete with the brush border binding of this light chain at all. This suggests that cubilin is the quantitatively major receptor for this λ-light chain. However, at maximal inhibitory concentration of the anti-cubilin antibody, 10% of light chain remained bound to brush border membranes, suggesting presence of additional binding sites for this light chain.

Anti-cubilin antiserum also inhibited endocytosis of light chain significantly. This further confirms that cubilin binding is followed by endocytosis of light chain. However, less than total inhibition of light chain endocytosis by anti-cubilin antibody indicates that this pathway may not be the exclusive endocytic pathway for light chains and that there may be alternate pathways which can compensate partially when the cubilin-mediated pathway is blocked. The antibodies used in the present study may be less than blocking functionally, and incomplete inhibition of endocytosis may be on this basis.

Importantly, binding of light chains to scavenger pathway receptors is not just a structural observation, as light chains had potent direct effects on endosomal fusion reconstituted in vitro. This raises the novel hypothesis that ligand binding may affect fusion properties of membranes, mediated by the receptors they bind. Select ligands are known to induce endocytosis of the ligand-receptor complex by binding, and the protein components of the final common pathway of fusion have largely been identified and cloned. These mechanisms may provide new insights into nephrotoxicity of myeloma light chains and other nephrotoxic low molecular weight proteins.

There are 13,500 new cases of myeloma annually in the U.S. and 1-4 new cases/100,000 of population worldwide. Although the precipitation of light chains with Tamm-Horsfall protein to form casts in renal distal nephron segments has been defined down to specific peptide sequences, the molecular characteristics of receptors that mediate the endocytosis of light chains in the proximal tubule have not been defined. Identification of the proximal tubular receptor for light chains extends and compliments these observations. The proximal tubule determines the distal delivery of low molecular weight proteins by reabsorbing the bulk of filtered proteins including light chains. Many low-molecular weight proteins induce injury to the proximal tubule, while others precipitate in the distal nephron. Both these mechanisms contribute to the pathogenesis of tubulointerstitial nephropathies associated with low-molecular-weight proteins, such as multiple myeloma. Proximal reabsorption of light chains is associated with tubular atrophy, necrosis and Fanconi syndrome. Taken together with understanding of distal tubular cast formation, identification of major renal binding proteins for myeloma light chains in the proximal tubules will allow detailed characterization of the binding site between cubilin and light chains, as well as other nephrotoxic low-molecular weight proteins. This completes the necessary mechanistic data of all affected nephron sites for the rational design of agents to protect from nephrotoxicity caused by myeloma light chains as well as other low-molecular weight proteins.

The present studies also demonstrate several lines of evidence suggesting that light chains are a ligand for megalin:

anti-megalin antiserum partially displaces brush border light chain binding, and gentamicin displaces brush border light chain binding. Independent evidence suggests that cubilin is a receptor for polybasic drugs as gentamicin directly interferes with light chain binding to cubilin in vitro. These observations are important, both to understand the complex interactions of toxic and physiological ligands on proximal tubule scavenger pathway receptors, as well as the eventual development of clinical protective agents for nephrotoxic damage mediated by ligands for cubilin and/or megalin.

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Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

40 1 11272 DNA rat nucleic acid sequence of rat cubilin 1 atgtcctcgc agtttctctg gggttttgtt actttgttga tgatagctga attagatggc 60 aaaactggaa agccggagca gaggggacag aaaaggatcg ctgacctgca ccagcctcgg 120 atgactacgg aggagggaaa cttagtattt cttacaagct ctacccaaaa cattgaattt 180 agaactggat ccctggggaa aatcaaatta aatgatgaag accttggcga atgtttacac 240 cagatccaga gaaacaaaga tgatattata gatttaagaa agaatacaac tggcctccct 300 caaaatatac ttagtcaagt ccaccaactt aactcgaagc tcgtggatct ggagagagat 360 tttcaaaact tacagcagaa tgtggagaga aaagtttgca gtagcaatcc ctgccttaat 420 ggtggcacct gcgtcaacct gcacgactcc tttgtgtgta tctgcccttc tcagtggaag 480 ggtctcttct gctcagagga tgtcaacgag tgtgtagttt actcaggaac accctttggc 540 tgccagagtg gatccacctg tgtgaacaca gtggggagtt tcagatgtga ctgcacgccg 600 gatacgtacg gacctcagtg tgcatccaaa tataatgact gtgaacaggg ctctaaacag 660 ctctgcaagc acggcatctg tgaggattta cagcgagttc accatggaca gccgaatttt 720 cactgcatct gtgatgctgg gtggacaaca ccgccaaatg gaatctcctg cacagaggac 780 aaagacgaat gcagcctcca gccttcacct tgttcggagc atgcgcagtg tttcaataca 840 caaggctcct tctactgtgg ggcctgcccc aaaggttggc agggaaatgg ttatgaatgc 900 caggatatca atgaatgcga gatcaacaat ggaggctgtt cccaggcgcc actggtccca 960 tgcctgaaca cacctggatc attctcctgt gggaactgtc cagcaggttt cagtggtgac 1020 gggagagtgt gcactcccgt ggacatctgt tcgatccaca atggaggctg ccatccagag 1080 gcaacctgct cctcatctcc tgttctaggg tccttcttgc ctgtctgcac ctgtcctccg 1140 ggatatacag gaaatggtta cgggtctaat ggatgtgtac gcctcagtaa tatttgctca 1200 aggcacccgt gtgtaaatgg acagtgcata gaaacagtat caagttattt ctgtaagtgt 1260 gactcaggct ggtctggaca gaactgtaca gaaaacatta atgactgttc gagcaacccc 1320 tgtttgaatg ggggcacctg tattgatggc atcaatggct tcacctgcga ctgcacaagc 1380 tcttggactg gctattactg tcagactccc caggcagctt gcggagggat cctctcaggg 1440 acacaaggaa cctttgccta ccacagccca aatgatacct atattcacaa tgtgaactgt 1500 ttctggattg tcagaactga tgaggaaaag gttctgcatg tcaccttcac gttttttgat 1560 ttagaatcag caagcaattg tccccgggag taccttcaga ttcatgatgg agactcctca 1620 gcggatttcc cacttggtag atactgtggc tccaggcccc cccaggggat ccacagcagt 1680 gccaatgctc tctatttcca tctctactcc gagtacataa ggagtgggag aggctttaca 1740 gcaaggtggg aggcgaagct gccagagtgt ggcggcatcc tgactgataa ttacggttct 1800 attacgtctc cggggtaccc tggaaactac cccccaggaa gagattgtgt ctggcaggtt 1860 ttagtcaatc ctaactcctt gataacattt acctttggaa ccttgagcct ggagagccac 1920 aatgactgca gcaaagacta tttggagatt cgagacggtc ctttccacca agaccctgtt 1980 cttgggaaat tctgcacttc cttgtctacc ccacccctca agactaccgg tcctgcagca 2040 agaattcatt tccattctga ctctgagacc agtgacaaag gcttccacat cacctatcta 2100 accacacagt cggatctgga ctgtggtggg aactacacag acacggatgg cgagctcctc 2160 cttcctcctt tgtctggtcc tttcagtcac agcagacagt gtgtctatct catcacccaa 2220 gcccagggag aacaaatagt tatcaacttc acccatgtgg agctggagag ccagatgggc 2280 tgttctcaca cttacatcga ggttggagac catgacagct tacttcgaaa gatctgtggc 2340 aatgaaacct tgttccccat tagatcagtt tctaataaag tctggatcag attgagaata 2400 gacgctttag tccagaaggc tagtttcaga gctgactacc aagttgcttg tgggggtatg 2460 ttaagaggag aaggattctt tcgctcacct ttctatccta acgcatatcc tggacgaaga 2520 acctgtaggt ggaccatctc ccaaccccaa agacaagttg tccttcttaa cttcactgac 2580 tttcagatcg gaagttctgc ctcctgtgat acagattata ttgagattgg tcccagctct 2640 gtcttgggat ctcctggaaa tgagaagttt tgtagctcaa acataccgtc atttataaca 2700 tctgtataca atattcttta tgttacattt gtgaaaagtt cttccatgga aaatcgtggc 2760 ttcacggcta agttcagcag tgacaaacta gagtgtggag aagttctcac ggcatctaca 2820 ggaattatcg aaagtccagg tcatccaaac gtctacccaa gaggtgtaaa ttgtacttgg 2880 catgtagtag tccaacgcgg ccaactgatc cgtttggagt tcagttcctt ttacctggag 2940 tttcattaca actgcacaaa cgactacctg gaaatttatg acactgccgc tcagactttt 3000 cttgggagat actgtggaaa atccatcccg ccttctctta ccagcaactc taattcaata 3060 aagctgatat ttgtgtctga ctccgccctt gcccacgaag gcttttccat aaattatgag 3120 gcaatcgatg catcatcagt atgtttatat gactatacag ataattttgg gatgctctcc 3180 tccccgaact tccccaataa ttaccccagt aactgggagt gcatctacag aatcactgtg 3240 ggactcaacc aacagattgc attgcatttc acagacttca ccttggagga ctattttggg 3300 tcacagtgtg tagattttgt agaaatcaga gacggaggct acgaaacgtc gccgcttgtt 3360 gggatttact gtggctcagt tttgcctcct acaatcatct cccacagtaa caagctctgg 3420 ctaaagttta agagtgacgc cgcactcacg gcaaaggggt tctcagcgta ctgggacgga 3480 tcatcaacag gctgtggagg taatctcacc accccacagg tgctcacatc gcccaactac 3540 ccgatgccct actaccacag ctccgaatgc tactggcggc tggaagccag tcatggcagc 3600 cctttcgagc tggaattcca agacttccac ctggaacacc accccagctg ctctctggat 3660 tacttgggcc gtgttgatgg cccgactacc aactcccgac tgatagataa attgtgtggg 3720 gatacgacac ctgctcccat ccgttccaat aaagacgtcg tattgttaaa aactgaggaa 3780 ctgatgcaag gtcagctagg ccgtggcttt gagatcaact tccggcagag atgtgacaat 3840 gtggtgatag tgaacaaaac ctttggcatc ctggagagca taaattatcc aaatccctat 3900 gataagaacc aacgttgtaa ctggaccatc caagcaacca ccggcaacac cgtgaactac 3960 acgtttctgg gatttgatgt ggaaagttac atgaactgct ccacagatta tgtagagctc 4020 tatgatggac cacaatggat gggacgctac tgtggaaata acatgccccc accaggggct 4080 acaacaggct cccaactcca cgtactgttc catacagatg ggatcaattc tggggaaaaa 4140 ggatttaaga tgcagtggtt cactcatggc tgtggtggag agatgtctgg aaccgcaggc 4200 tccttcagca gccctgggta ccccaacagc tatcctcaca acaaagagtg tatctggaac 4260 attcgcgtgg ccccagggag tagcattcag ctcaccatcc atgactttga tgtggaatat 4320 catacaagct gcaactatga ctccctggag atctatgcag gtcttgattt taactctcca 4380 agaatagccc aactgtgttc ccaatcaccg tcagcgaacc ccatgcaggt ctccagcact 4440 ggcaatgaac tagcaatccg atttaagacg gatagcactt taaatggaag aggtttcaat 4500 gcctcgtggc gagcagtccc tggaggttgt ggtggaatta tccagctttc cagaggagag 4560 attcattctc caaattaccc caacaactac agagctaaca cagagtgctc ctggatcatt 4620 caagttgagc gacatcaccg tgttctcttg aatatcactg actttgacct tgaagctcca 4680 gattcttgct tacgacttat ggatggctca agttccacaa acgcccgtgt cgccagtgtg 4740 tgtggaagac agcagccccc taactctatc atcgcttcag gaaacagcct ctttgtgaga 4800 ttccggtctg gatcttccag ccagaacagg ggcttccggg ctgaattcag ggaagagtgc 4860 ggaggccgca tcatgaccga ctcttccgat actatcttct ctccactgta ccctcacaac 4920 tatctacaca accagaactg ttcctggata attgaagctc agcctccatt caatcacatt 4980 actctctcct ttactcactt tcaacttcaa aacagcacag actgtacacg ggactttgta 5040 gaaattttgg atggcaacga ctatgacgca cctgtccaag gccgttactg tggtttctcc 5100 ctgccccacc ccatcatatc atttggcaat gccctaaccg tgaggtttgt cactgattcc 5160 acacgcagtt ttgagggttt ccgtgccatc tattctgcat cgacatcatc ttgtggtgga 5220 agcttctaca cacttgatgg catcttcaat agccccgact acccagcaga ctaccatcca 5280 aatgcagaat gtgtctggaa cattgccagc tcccctggca accgcctgca actgtccttc 5340 ctatccttca atttggagaa ttctctaaac tgtaacaagg attttgtgga aatccgagaa 5400 ggaaatgcca cgggccactt gattggacga tactgtggaa actccctccc tgggaattat 5460 tcgtcagctg agggacatag tctatgggtc cgatttgtct ctgatggctc aggcactggc 5520 atgggcttcc aggccaggtt caaaaatata tttggcaata ataatattgt gggaactcat 5580 gggaaaatcg catctccctt ctggcctgga aaatacccct acaactccaa ttacaaatgg 5640 gtggtaaatg tggacgcata tcatattatc cacggtagaa tcttagagat ggacatagaa 5700 cccacaacga actgctttta tgacagttta aagatttatg atggatttga cactcattcc 5760 cgtctcattg gcacttactg tggtacccag acagaatcct ttagctccag tagaaactat 5820 ctgacattcc agttttcttc cgactcttct gtgtcaggaa ggggattcct tctggagtgg 5880 tttgcagtag atgtttctga tagcacccct cccaccatcg ctccaggagc ttgtggaggg 5940 tttatggtga cgggagacac tcctgtccat attttctccc cgggttggcc tagagagtat 6000 gctaatggtg ctgactgtat ctggatcatc tatgctcctg actctactgt ggaactcaac 6060 attctctcct tggacatcga accgcagcag tcatgcaatt atgacaagct gatcgtaaaa 6120 gacggagaca gtgacttatc cccagagctg gctgttctgt gtggcgtaag ccctcctggg 6180 cccatccggt caactggaga atacatgtac atccgcttca cttcagacac cagcgtcgcg 6240 gggacaggct tcaacgcctc ctttcacaag agctgtggtg gatatttgca tgcagatcga 6300 ggagttatca catcccccaa gtatccagac acctaccttc ccaacctcaa ctgctcctgg 6360 catgttctgg tccagactgg tctgaccatc gccgtccatt ttgagcagcc tttccagatt 6420 caaaacagag actctttttg cagtcagggg gattacttgg tgctaagaaa cggaccagat 6480 aaccattctc caccactggg accttctgga agaaatggtc gtttctgcgg aatgtacgca 6540 ccgtccactc tgttcacctc aggcaatgaa atgtttgttc agttcatctc ggacagtagt 6600 aatggtggac aagggtttaa gatcagatat gaggcaaaga gtttagcctg cgggggcact 6660 gtctacatcc atgatgctga ctctgacgga tacctgacct cccccaacta ccctgctaat 6720 tatccccaac atgccgaatg catttggatc ttagaggcgc ctccagggag aagcatacag 6780 ctccaatttg aagatcaatt caatattgaa gacacaccca actgttctgt gagctatctt 6840 gaattgcgtg acggagccaa ctcgaatgca cgtctggttt ccaagttgtg tggccacact 6900 ctgcctcata gctgggtatc ctcgagagaa cgaatatact tgaagtttca cactgacggt 6960 ggttccagct acatgggatt caaggccaag tactctatag cttcctgtgg aggaacagtc 7020 tcaggggaca gtggagtcat cgagagcatt ggctacccga cccttccgta tgcaaacaat 7080 gtgttttgtc agtggtttat ccgaggcctc ccaggacact acctcactct cagttttgaa 7140 gattttaacc ttcagagctc tcctggttgt acaaaagact ttgtggagat ctgggaaaac 7200 catacctctg gaagagttct ggggagatat tgtggaaact ccactcctag cagtgttgac 7260 acttccagca atgttgcttc cgtcaagttt gtcacagatg gctctgtcac tgcctcagga 7320 tttaggctgc agtttaagtc cagcagacaa gtgtgtggtg gggatttaca tggccctact 7380 ggcacattta cttctcccaa ctacccaaac ccaaatcctc atgcccggat ctgtgagtgg 7440 acgatcactg tacaagaagg aaggcggatc gtcctgacgt ttaccaactt gaggctgagt 7500 acccagccat cttgtaacag tgagcacctc atcgtattca atggcattag aagcaactcg 7560 cccctactac agaaactgtg cagccgtgtg aatgtgacca atgaattcaa atcttcagga 7620 aacaccatga aagtggtatt tttcactgat ggctcccggc cgtatggagg cttcactgct 7680 tcctacacct ctactgaaga tgcagtgtgt ggtggatttc ttccaagtgt ctccggtgga 7740 aacttttctt ctcctggcta taatggaatc cgtgattatg ccagaaacct agactgtgaa 7800 tggactctca gtaatccaaa tcgggaaaat tcatccataa gtatctattt tctagaactt 7860 tccattgaaa gtcatcaaga ctgtacattt gatgtccttg agtttcgagt aggggatgct 7920 gatgggcccc tgatagagaa gttctgtagc ctgtcagcac caacagcgcc cttggtcatc 7980 ccctaccctc aggtgtggat acgcttcgtc agcaatgagc gtgtagaata tactggattc 8040 tatatagagt actcctttac agattgtggt ggaatacgga caggtgacaa tggagtgatc 8100 tcaagtccta actatccaaa cttgtacagt gcatggaccc actgttcatg gctgctgaaa 8160 gccccagaag ggcacaccat cactctcaca ctcagtgact ttcttctcga ggctcatcca 8220 acttgcactt cagactccgt cactgtcagg aatggtgact ccccaggatc gcccgtcata 8280 ggacgatact gtggacagtc agtcccaagg ccgatacagt ctggttccaa ccaacttata 8340 gtgactttta acacaaacaa tcaagggcaa actcgtggat tttatgcaac atggaccaca 8400 aacgctttag gttgtggggg aacattccac tcagctaatg gtacaatcaa atctcctcac 8460 tggcctcaga cattcccaga aaacagcaga tgctcctgga cagtgatcac tcacgatagt 8520 aaacactggg agattagctt tgacagcaat ttccgaatcc ccagcagtga cagccagtgt 8580 cagaacagct tcgtgaaggt ttggggaggc aggttgatga tcaataagac cctgttagcc 8640 acgagctgtg gagatgtggc tccaagtccc attgtcacat cagggaacat tttcactgct 8700 gtcttccaat ctgaggagat ggcagcccag ggcttctctg catccttcat tagccggtgc 8760 ggacgcacat tcaatacctc ccctggtgac atcatctctc caaacttccc gaagcaatac 8820 gacaacaaca tgaactgcac ctacctcata gacgctgacc ctcagtctct ggtcatcctg 8880 acttttgtgt cctttcattt ggaagatcgc tcagctatca ccggaacctg tgatcatgat 8940 ggcttgcaca tcatcaaagg tcgtaacctc tcttccactc ctctcgtgac catatgtggt 9000 tctgaaactt tgcgtcccct cactgtggac ggcccagtgt tgctcaactt ctattctgat 9060 gcatacacca cagactttgg cttcaagatt tcctacagag ccatcacctg tggtgggatc 9120 tacaatgaat cctctggaat ccttaggagc ccttcctact catacagcaa ctaccccaac 9180 aacctctact gtgtctacag cctccatgtt agaagcagca gagtgataat aattaggttc 9240 aatgatttcg atgtggctcc ttccaacctt tgtgcacatg acttcctgga ggtgtttgat 9300 ggtcccagca ttggaaatcg atctcttgga aagttctgtg gttccacgcg tccacaaact 9360 gttaagagca ccaatagcag cctgaccctg ctgttcaaga cagattcttc tcaaacagca 9420 agaggttgga aaatattttt ccgggagaca atagggccac agcagggatg tggtggatac 9480 ctgaccgagg acaaccagag ctttgtgtct cctgattctg attcgaatgg acgctatgac 9540 aagggtctca gctgcatatg gtacatagtt gcacctgaaa acaaactggt taagctcacc 9600 ttcaatgtgt tcactctgga gggaccatcg tcagctggga gctgcgtcta tgattatgtg 9660 cagatagcag atggcgcaag cataaactca tatttaggtg gaaaattctg tggctcccgt 9720 atgcctgccc catttatctc ttccggctac ttccttacgt ttcagtttgt ctctgacgta 9780 actgttgaaa tgaggggatt taatgcaaca tatacctttg tggacatgcc ttgcggggga 9840 acatataacg caacctcgac acctcaaaat gcgtcatcac ctcatttatc caacatcgga 9900 cggccatact ccacctgtac ttgggtcatc gcagctcccc cacagcagca ggttcagata 9960 actgtgtggg acttacagct gccctcacaa gactgctcac aaagctactt agaacttcag 10020 gattcagtac agactggtgg aaaccgggtg actcagttct gcggtgcgaa ctatacaacc 10080 ttgccagtgt tctactcctc aatgagcact gcggttgtcg ttttcaagtc tggagttata 10140 aacagaaact cgcaagtgca attctcctat cagattgcag attgcaacag agaatacaac 10200 caaacgtttg gcaatctgaa gagtcctggg tggcctcaga actatgacaa taacctggac 10260 tgcaccatca ttctcagagc cccgcagaac cacagcattt ccctcttttt ctattggttt 10320 cagctggaag attcaagaca atgcatgaat gatttcttgg aggtaagaaa cggcggcagc 10380 agcacctcac cactgcttga caagtactgt agcaacctgc tgcccaaccc ggtcttctct 10440 cagagcaacg aactgtatct gcactttcac agcgaccact cagtcaccaa caatggctat 10500 gaaattattt ggacctcctc tgctgctggc tgtggaggga ctcttttggg cgacgaaggg 10560 atattcacca accctggctt tcctgacagt tacccgaaca acactcattg tgaatggacc 10620 attgttgctc cttctggaag gcctgtctct gtcgggtttc cctttctcag tatcgactct 10680 tctggtggct gtgaccagaa ctacctcata gtctttaatg gtccagacgc caactcccca 10740 ccctttggac cgttgtgtgg catcaacact gggatagcgc ccttctatgc ttcatcaaat 10800 cgggtcttta taaggtttca tgctgagtat acgacacgtc tttcagggtt tgaaataatg 10860 tggagcagct gaatcaggag agctgtgtat aaccccagga ctctggttct gcccagtgct 10920 gtcagatgca accctgccca gtactcattc tgctgttctc atccccgtct ctgcctgcgt 10980 catcaaacat ggactgagct tctacagcct tgaccagaga aagtgcacga ctgacttcat 11040 acattaggcg ttctgaaggc tttgatctac tcagctgtgc acaccatctt tactggatgc 11100 cctgtttagc cgacatcttc taaaatgttc tttaaagggt ggaagttatc ctggcacatt 11160 gatgtacggt tttaaaactt ggtgatacaa atggatgtat tgttccacta caaaagtcaa 11220 agtgcaggta ataaacattc ttcacatgta aaaaaaaaaa aaaaaaaaaa aa 11272 2 3623 PRT rat amino acid sequence of rat cubilin protein 2 Met Ser Ser Gln Phe Leu Trp Gly Phe Val Thr Leu Leu Met Ile 5 10 15 Ala Glu Leu Asp Gly Lys Thr Gly Lys Pro Glu Gln Arg Gly Gln 20 25 30 Lys Arg Ile Ala Asp Leu Gly Gln Pro Arg Met Thr Thr Glu Glu 35 40 45 Gly Asn Leu Val Phe Leu Thr Ser Ser Thr Gln Asn Ile Glu Phe 50 55 60 Arg Thr Gly Ser Leu Gly Lys Ile Lys Leu Asn Asp Glu Asp Leu 65 70 75 Gly Glu Cys Leu Gly Gln Ile Gln Arg Asn Lys Asp Asp Ile Ile 80 85 90 Asp Leu Arg Lys Asn Thr Thr Gly Leu Pro Gln Asn Ile Leu Ser 95 100 105 Gln Val Gly Gln Leu Asn Ser Lys Leu Val Asp Leu Glu Arg Asp 110 115 120 Phe Gln Asn Leu Gln Gln Asn Val Glu Arg Lys Val Cys Ser Ser 125 130 135 Asn Pro Cys Leu Asn Gly Gly Thr Cys Val Asn Leu Gly Asp Ser 140 145 150 Phe Val Cys Ile Cys Pro Ser Gln Trp Lys Gly Leu Phe Cys Ser 155 160 165 Glu Asp Val Asn Glu Cys Val Val Tyr Ser Gly Thr Pro Phe Gly 170 175 180 Cys Gln Ser Gly Ser Thr Cys Val Asn Thr Val Gly Ser Phe Arg 185 190 195 Cys Asp Cys Thr Pro Asp Thr Tyr Gly Pro Gln Cys Ala Ser Lys 200 205 210 Tyr Asn Asp Cys Glu Gln Gly Ser Lys Gln Leu Cys Lys Gly Gly 215 220 225 Ile Cys Glu Asp Leu Gln Arg Val Gly Gly Gly Gln Pro Asn Phe 230 235 240 Gly Cys Ile Cys Asp Ala Gly Trp Thr Thr Pro Pro Asn Gly Ile 245 250 255 Ser Cys Thr Glu Asp Lys Asp Glu Cys Ser Leu Gln Pro Ser Pro 260 265 270 Cys Ser Glu Gly Ala Gln Cys Phe Asn Thr Gln Gly Ser Phe Tyr 275 280 285 Cys Gly Ala Cys Pro Lys Gly Trp Gln Gly Asn Gly Tyr Glu Cys 290 295 300 Gln Asp Ile Asn Glu Cys Glu Ile Asn Asn Gly Gly Cys Ser Gln 305 310 315 Ala Pro Leu Val Pro Cys Leu Asn Thr Pro Gly Ser Phe Ser Cys 320 325 330 Gly Asn Cys Pro Ala Gly Phe Ser Gly Asp Gly Arg Val Cys Thr 335 340 345 Pro Val Asp Ile Cys Ser Ile Gly Asn Gly Gly Cys Gly Pro Glu 350 355 360 Ala Thr Cys Ser Ser Ser Pro Val Leu Gly Ser Phe Leu Pro Val 365 370 375 Cys Thr Cys Pro Pro Gly Tyr Thr Gly Asn Gly Tyr Gly Ser Asn 380 385 390 Gly Cys Val Arg Leu Ser Asn Ile Cys Ser Arg Gly Pro Cys Val 395 400 405 Asn Gly Gln Cys Ile Glu Thr Val Ser Ser Tyr Phe Cys Lys Cys 410 415 420 Asp Ser Gly Trp Ser Gly Gln Asn Cys Thr Glu Asn Ile Asn Asp 425 430 435 Cys Ser Ser Asn Pro Cys Leu Asn Gly Gly Thr Cys Ile Asp Gly 440 445 450 Ile Asn Gly Phe Thr Cys Asp Cys Thr Ser Ser Trp Thr Gly Tyr 455 460 465 Tyr Cys Gln Thr Pro Gln Ala Ala Cys Gly Gly Ile Leu Ser Gly 470 475 480 Thr Gln Gly Thr Phe Ala Tyr Gly Ser Pro Asn Asp Thr Tyr Ile 485 490 495 Gly Asn Val Asn Cys Phe Trp Ile Val Arg Thr Asp Glu Glu Lys 500 505 510 Val Leu Gly Val Thr Phe Thr Phe Phe Asp Leu Glu Ser Ala Ser 515 520 525 Asn Cys Pro Arg Glu Tyr Leu Gln Ile Gly Asp Gly Asp Ser Ser 530 535 540 Ala Asp Phe Pro Leu Gly Arg Tyr Cys Gly Ser Arg Pro Pro Gln 545 550 555 Gly Ile Gly Ser Ser Ala Asn Ala Leu Tyr Phe Gly Leu Tyr Ser 560 565 570 Glu Tyr Ile Arg Ser Gly Arg Gly Phe Thr Ala Arg Trp Glu Ala 575 580 585 Lys Leu Pro Glu Cys Gly Gly Ile Leu Thr Asp Asn Tyr Gly Ser 590 595 600 Ile Thr Ser Pro Gly Tyr Pro Gly Asn Tyr Pro Pro Gly Arg Asp 605 610 615 Cys Val Trp Gln Val Leu Val Asn Pro Asn Ser Leu Ile Thr Phe 620 625 630 Thr Phe Gly Thr Leu Ser Leu Glu Ser Gly Asn Asp Cys Ser Lys 635 640 645 Asp Tyr Leu Glu Ile Arg Asp Gly Pro Phe Gly Gln Asp Pro Val 650 655 660 Leu Gly Lys Phe Cys Thr Ser Leu Ser Thr Pro Pro Leu Lys Thr 665 670 675 Thr Gly Pro Ala Ala Arg Ile Gly Phe Gly Ser Asp Ser Glu Thr 680 685 690 Ser Asp Lys Gly Phe Gly Ile Thr Tyr Leu Thr Thr Gln Ser Asp 695 700 705 Leu Asp Cys Gly Gly Asn Tyr Thr Asp Thr Asp Gly Glu Leu Leu 710 715 720 Leu Pro Pro Leu Ser Gly Pro Phe Ser Gly Ser Arg Gln Cys Val 725 730 735 Tyr Leu Ile Thr Gln Ala Gln Gly Glu Gln Ile Val Ile Asn Phe 740 745 750 Thr Gly Val Glu Leu Glu Ser Gln Met Gly Cys Ser Gly Thr Tyr 755 760 765 Ile Glu Val Gly Asp Gly Asp Ser Leu Leu Arg Lys Ile Cys Gly 770 775 780 Asn Glu Thr Leu Phe Pro Ile Arg Ser Val Ser Asn Lys Val Trp 785 790 795 Ile Arg Leu Arg Ile Asp Ala Leu Val Gln Lys Ala Ser Phe Arg 800 805 810 Ala Asp Tyr Gln Val Ala Cys Gly Gly Met Leu Arg Gly Glu Gly 815 820 825 Phe Phe Arg Ser Pro Phe Tyr Pro Asn Ala Tyr Pro Gly Arg Arg 830 835 840 Thr Cys Arg Trp Thr Ile Ser Gln Pro Gln Arg Gln Val Val Leu 845 850 855 Leu Asn Phe Thr Asp Phe Gln Ile Gly Ser Ser Ala Ser Cys Asp 860 865 870 Thr Asp Tyr Ile Glu Ile Gly Pro Ser Ser Val Leu Gly Ser Pro 875 880 885 Gly Asn Glu Lys Phe Cys Ser Ser Asn Ile Pro Ser Phe Ile Thr 890 895 900 Ser Val Tyr Asn Ile Leu Tyr Val Thr Phe Val Lys Ser Ser Ser 905 910 915 Met Glu Asn Arg Gly Phe Thr Ala Lys Phe Ser Ser Asp Lys Leu 920 925 930 Glu Cys Gly Glu Val Leu Thr Ala Ser Thr Gly Ile Ile Glu Ser 935 940 945 Pro Gly Gly Pro Asn Val Tyr Pro Arg Gly Val Asn Cys Thr Trp 950 955 960 Gly Val Val Val Gln Arg Gly Gln Leu Ile Arg Leu Glu Phe Ser 965 970 975 Ser Phe Tyr Leu Glu Phe Gly Tyr Asn Cys Thr Asn Asp Tyr Leu 980 985 990 Glu Ile Tyr Asp Thr Ala Ala Gln Thr Phe Leu Gly Arg Tyr Cys 995 1000 1005 Gly Lys Ser Ile Pro Pro Ser Leu Thr Ser Asn Ser Asn Ser Ile 1010 1015 1020 Lys Leu Ile Phe Val Ser Asp Ser Ala Leu Ala Gly Glu Gly Phe 1025 1030 1035 Ser Ile Asn Tyr Glu Ala Ile Asp Ala Ser Ser Val Cys Leu Tyr 1040 1045 1050 Asp Tyr Thr Asp Asn Phe Gly Met Leu Ser Ser Pro Asn Phe Pro 1055 1060 1065 Asn Asn Tyr Pro Ser Asn Trp Glu Cys Ile Tyr Arg Ile Thr Val 1070 1075 1080 Gly Leu Asn Gln Gln Ile Ala Leu Gly Phe Thr Asp Phe Thr Leu 1085 1090 1095 Glu Asp Tyr Phe Gly Ser Gln Cys Val Asp Phe Val Glu Ile Arg 1100 1105 1110 Asp Gly Gly Tyr Glu Thr Ser Pro Leu Val Gly Ile Tyr Cys Gly 1115 1120 1125 Ser Val Leu Pro Pro Thr Ile Ile Ser Gly Ser Asn Lys Leu Trp 1130 1135 1140 Leu Lys Phe Lys Ser Asp Ala Ala Leu Thr Ala Lys Gly Phe Ser 1145 1150 1155 Ala Tyr Trp Asp Gly Ser Ser Thr Gly Cys Gly Gly Asn Leu Thr 1160 1165 1170 Thr Pro Gln Val Leu Thr Ser Pro Asn Tyr Pro Met Pro Tyr Tyr 1175 1180 1185 Gly Ser Ser Glu Cys Tyr Trp Arg Leu Glu Ala Ser Gly Gly Ser 1190 1195 1200 Pro Phe Glu Leu Glu Phe Gln Asp Phe Gly Leu Glu Gly Gly Pro 1205 1210 1215 Ser Cys Ser Leu Asp Tyr Leu Gly Arg Val Asp Gly Pro Thr Thr 1220 1225 1230 Asn Ser Arg Leu Ile Asp Lys Leu Cys Gly Asp Thr Thr Pro Ala 1235 1240 1245 Pro Ile Arg Ser Asn Lys Asp Val Val Leu Leu Lys Thr Glu Glu 1250 1255 1260 Leu Met Gln Gly Gln Leu Gly Arg Gly Phe Glu Ile Asn Phe Arg 1265 1270 1275 Gln Arg Cys Asp Asn Val Val Ile Val Asn Lys Thr Phe Gly Ile 1280 1285 1290 Leu Glu Ser Ile Asn Tyr Pro Asn Pro Tyr Asp Lys Asn Gln Arg 1295 1300 1305 Cys Asn Trp Thr Ile Gln Ala Thr Thr Gly Asn Thr Val Asn Tyr 1310 1315 1320 Thr Phe Leu Gly Phe Asp Val Glu Ser Tyr Met Asn Cys Ser Thr 1325 1330 1335 Asp Tyr Val Glu Leu Tyr Asp Gly Pro Gln Trp Met Gly Arg Tyr 1340 1345 1350 Cys Gly Asn Asn Met Pro Pro Pro Gly Ala Thr Thr Gly Ser Gln 1355 1360 1365 Leu Gly Val Leu Phe Gly Thr Asp Gly Ile Asn Ser Gly Glu Lys 1370 1375 1380 Gly Phe Lys Met Gln Trp Phe Thr Gly Gly Cys Gly Gly Glu Met 1385 1390 1395 Ser Gly Thr Ala Gly Ser Phe Ser Ser Pro Gly Tyr Pro Asn Ser 1400 1405 1410 Tyr Pro Gly Asn Lys Glu Cys Ile Trp Asn Ile Arg Val Ala Pro 1415 1420 1425 Gly Ser Ser Ile Gln Leu Thr Ile Gly Asp Phe Asp Val Glu Tyr 1430 1435 1440 Gly Thr Ser Cys Asn Tyr Asp Ser Leu Glu Ile Tyr Ala Gly Leu 1445 1450 1455 Asp Phe Asn Ser Pro Arg Ile Ala Gln Leu Cys Ser Gln Ser Pro 1460 1465 1470 Ser Ala Asn Pro Met Gln Val Ser Ser Thr Gly Asn Glu Leu Ala 1475 1480 1485 Ile Arg Phe Lys Thr Asp Ser Thr Leu Asn Gly Arg Gly Phe Asn 1490 1495 1500 Ala Ser Trp Arg Ala Val Pro Gly Gly Cys Gly Gly Ile Ile Gln 1505 1510 1515 Leu Ser Arg Gly Glu Ile Gly Ser Pro Asn Tyr Pro Asn Asn Tyr 1520 1525 1530 Arg Ala Asn Thr Glu Cys Ser Trp Ile Ile Gln Val Glu Arg Gly 1535 1540 1545 Gly Arg Val Leu Leu Asn Ile Thr Asp Phe Asp Leu Glu Ala Pro 1550 1555 1560 Asp Ser Cys Leu Arg Leu Met Asp Gly Ser Ser Ser Thr Asn Ala 1565 1570 1575 Arg Val Ala Ser Val Cys Gly Arg Gln Gln Pro Pro Asn Ser Ile 1580 1585 1590 Ile Ala Ser Gly Asn Ser Leu Phe Val Arg Phe Arg Ser Gly Ser 1595 1600 1605 Ser Ser Gln Asn Arg Gly Phe Arg Ala Glu Phe Arg Glu Glu Cys 1610 1615 1620 Gly Gly Arg Ile Met Thr Asp Ser Ser Asp Thr Ile Phe Ser Pro 1625 1630 1635 Leu Tyr Pro Gly Asn Tyr Leu Gly Asn Gln Asn Cys Ser Trp Ile 1640 1645 1650 Ile Glu Ala Gln Pro Pro Phe Asn Gly Ile Thr Leu Ser Phe Thr 1655 1660 1665 Gly Phe Gln Leu Gln Asn Ser Thr Asp Cys Thr Arg Asp Phe Val 1670 1675 1680 Glu Ile Leu Asp Gly Asn Asp Tyr Asp Ala Pro Val Gln Gly Arg 1685 1690 1695 Tyr Cys Gly Phe Ser Leu Pro Gly Pro Ile Ile Ser Phe Gly Asn 1700 1705 1710 Ala Leu Thr Val Arg Phe Val Thr Asp Ser Thr Arg Ser Phe Glu 1715 1720 1725 Gly Phe Arg Ala Ile Tyr Ser Ala Ser Thr Ser Ser Cys Gly Gly 1730 1735 1740 Ser Phe Tyr Thr Leu Asp Gly Ile Phe Asn Ser Pro Asp Tyr Pro 1745 1750 1755 Ala Asp Tyr Gly Pro Asn Ala Glu Cys Val Trp Asn Ile Ala Ser 1760 1765 1770 Ser Pro Gly Asn Arg Leu Gln Leu Ser Phe Leu Ser Phe Asn Leu 1775 1780 1785 Glu Asn Ser Leu Asn Cys Asn Lys Asp Phe Val Glu Ile Arg Glu 1790 1795 1800 Gly Asn Ala Thr Gly Gly Leu Ile Gly Arg Tyr Cys Gly Asn Ser 1805 1810 1815 Leu Pro Gly Asn Tyr Ser Ser Ala Glu Gly Gly Ser Leu Trp Val 1820 1825 1830 Arg Phe Val Ser Asp Gly Ser Gly Thr Gly Met Gly Phe Gln Ala 1835 1840 1845 Arg Phe Lys Asn Ile Phe Gly Asn Asn Asn Ile Val Gly Thr Gly 1850 1855 1860 Gly Lys Ile Ala Ser Pro Phe Trp Pro Gly Lys Tyr Pro Tyr Asn 1865 1870 1875 Ser Asn Tyr Lys Trp Val Val Asn Val Asp Ala Tyr Gly Ile Ile 1880 1885 1890 Gly Gly Arg Ile Leu Glu Met Asp Ile Glu Pro Thr Thr Asn Cys 1895 1900 1905 Phe Tyr Asp Ser Leu Lys Ile Tyr Asp Gly Phe Asp Thr Gly Ser 1910 1915 1920 Arg Leu Ile Gly Thr Tyr Cys Gly Thr Gln Thr Glu Ser Phe Ser 1925 1930 1935 Ser Ser Arg Asn Tyr Leu Thr Phe Gln Phe Ser Ser Asp Ser Ser 1940 1945 1950 Val Ser Gly Arg Gly Phe Leu Leu Glu Trp Phe Ala Val Asp Val 1955 1960 1965 Ser Asp Ser Thr Pro Pro Thr Ile Ala Pro Gly Ala Cys Gly Gly 1970 1975 1980 Phe Met Val Thr Gly Asp Thr Pro Val Gly Ile Phe Ser Pro Gly 1985 1990 1995 Trp Pro Arg Glu Tyr Ala Asn Gly Ala Asp Cys Ile Trp Ile Ile 2000 2005 2010 Tyr Ala Pro Asp Ser Thr Val Glu Leu Asn Ile Leu Ser Leu Asp 2015 2020 2025 Ile Glu Pro Gln Gln Ser Cys Asn Tyr Asp Lys Leu Ile Val Lys 2030 2035 2040 Asp Gly Asp Ser Asp Leu Ser Pro Glu Leu Ala Val Leu Cys Gly 2045 2050 2055 Val Ser Pro Pro Gly Pro Ile Arg Ser Thr Gly Glu Tyr Met Tyr 2060 2065 2070 Ile Arg Phe Thr Ser Asp Thr Ser Val Ala Gly Thr Gly Phe Asn 2075 2080 2085 Ala Ser Phe Gly Lys Ser Cys Gly Gly Tyr Leu Gly Ala Asp Arg 2090 2095 2100 Gly Val Ile Thr Ser Pro Lys Tyr Pro Asp Thr Tyr Leu Pro Asn 2105 2110 2115 Leu Asn Cys Ser Trp Gly Val Leu Val Gln Thr Gly Leu Thr Ile 2120 2125 2130 Ala Val Gly Phe Glu Gln Pro Phe Gln Ile Gln Asn Arg Asp Ser 2135 2140 2145 Phe Cys Ser Gln Gly Asp Tyr Leu Val Leu Arg Asn Gly Pro Asp 2150 2155 2160 Asn Gly Ser Pro Pro Leu Gly Pro Ser Gly Arg Asn Gly Arg Phe 2165 2170 2175 Cys Gly Met Tyr Ala Pro Ser Thr Leu Phe Thr Ser Gly Asn Glu 2180 2185 2190 Met Phe Val Gln Phe Ile Ser Asp Ser Ser Asn Gly Gly Gln Gly 2195 2200 2205 Phe Lys Ile Arg Tyr Glu Ala Lys Ser Leu Ala Cys Gly Gly Thr 2210 2215 2220 Val Tyr Ile Gly Asp Ala Asp Ser Asp Gly Tyr Leu Thr Ser Pro 2225 2230 2235 Asn Tyr Pro Ala Asn Tyr Pro Gln Gly Ala Glu Cys Ile Trp Ile 2240 2245 2250 Leu Glu Ala Pro Pro Gly Arg Ser Ile Gln Leu Gln Phe Glu Asp 2255 2260 2265 Gln Phe Asn Ile Glu Asp Thr Pro Asn Cys Ser Val Ser Tyr Leu 2270 2275 2280 Glu Leu Arg Asp Gly Ala Asn Ser Asn Ala Arg Leu Val Ser Lys 2285 2290 2295 Leu Cys Gly Gly Thr Leu Pro Gly Ser Trp Val Ser Ser Arg Glu 2300 2305 2310 Arg Ile Tyr Leu Lys Phe Gly Thr Asp Gly Gly Ser Ser Tyr Met 2315 2320 2325 Gly Phe Lys Ala Lys Tyr Ser Ile Ala Ser Cys Gly Gly Thr Val 2330 2335 2340 Ser Gly Asp Ser Gly Val Ile Glu Ser Ile Gly Tyr Pro Thr Leu 2345 2350 2355 Pro Tyr Ala Asn Asn Val Phe Cys Gln Trp Phe Ile Arg Gly Leu 2360 2365 2370 Pro Gly Gly Tyr Leu Thr Leu Ser Phe Glu Asp Phe Asn Leu Gln 2375 2380 2385 Ser Ser Pro Gly Cys Thr Lys Asp Phe Val Glu Ile Trp Glu Asn 2390 2395 2400 Gly Thr Ser Gly Arg Val Leu Gly Arg Tyr Cys Gly Asn Ser Thr 2405 2410 2415 Pro Ser Ser Val Asp Thr Ser Ser Asn Val Ala Ser Val Lys Phe 2420 2425 2430 Val Thr Asp Gly Ser Val Thr Ala Ser Gly Phe Arg Leu Gln Phe 2435 2440 2445 Lys Ser Ser Arg Gln Val Cys Gly Gly Asp Leu Gly Gly Pro Thr 2450 2455 2460 Gly Thr Phe Thr Ser Pro Asn Tyr Pro Asn Pro Asn Pro Gly Ala 2465 2470 2475 Arg Ile Cys Glu Trp Thr Ile Thr Val Gln Glu Gly Arg Arg Ile 2480 2485 2490 Val Leu Thr Phe Thr Asn Leu Arg Leu Ser Thr Gln Pro Ser Cys 2495 2500 2505 Asn Ser Glu Gly Leu Ile Val Phe Asn Gly Ile Arg Ser Asn Ser 2510 2515 2520 Pro Leu Leu Gln Lys Leu Cys Ser Arg Val Asn Val Thr Asn Glu 2525 2530 2535 Phe Lys Ser Ser Gly Asn Thr Met Lys Val Val Phe Phe Thr Asp 2540 2545 2550 Gly Ser Arg Pro Tyr Gly Gly Phe Thr Ala Ser Tyr Thr Ser Thr 2555 2560 2565 Glu Asp Ala Val Cys Gly Gly Phe Leu Pro Ser Val Ser Gly Gly 2570 2575 2580 Asn Phe Ser Ser Pro Gly Tyr Asn Gly Ile Arg Asp Tyr Ala Arg 2585 2590 2595 Asn Leu Asp Cys Glu Trp Thr Leu Ser Asn Pro Asn Arg Glu Asn 2600 2605 2610 Ser Ser Ile Ser Ile Tyr Phe Leu Glu Leu Ser Ile Glu Ser Gly 2615 2620 2625 Gln Asp Cys Thr Phe Asp Val Leu Glu Phe Arg Val Gly Asp Ala 2630 2635 2640 Asp Gly Pro Leu Ile Glu Lys Phe Cys Ser Leu Ser Ala Pro Thr 2645 2650 2655 Ala Pro Leu Val Ile Pro Tyr Pro Gln Val Trp Ile Arg Phe Val 2660 2665 2670 Ser Asn Glu Arg Val Glu Tyr Thr Gly Phe Tyr Ile Glu Tyr Ser 2675 2680 2685 Phe Thr Asp Cys Gly Gly Ile Arg Thr Gly Asp Asn Gly Val Ile 2690 2695 2700 Ser Ser Pro Asn Tyr Pro Asn Leu Tyr Ser Ala Trp Thr Gly Cys 2705 2710 2715 Ser Trp Leu Leu Lys Ala Pro Glu Gly Gly Thr Ile Thr Leu Thr 2720 2725 2730 Leu Ser Asp Phe Leu Leu Glu Ala Gly Pro Thr Cys Thr Ser Asp 2735 2740 2745 Ser Val Thr Val Arg Asn Gly Asp Ser Pro Gly Ser Pro Val Ile 2750 2755 2760 Gly Arg Tyr Cys Gly Gln Ser Val Pro Arg Pro Ile Gln Ser Gly 2765 2770 2775 Ser Asn Gln Leu Ile Val Thr Phe Asn Thr Asn Asn Gln Gly Gln 2780 2785 2790 Thr Arg Gly Phe Tyr Ala Thr Trp Thr Thr Asn Ala Leu Gly Cys 2795 2800 2805 Gly Gly Thr Phe Gly Ser Ala Asn Gly Thr Ile Lys Ser Pro Gly 2810 2815 2820 Trp Pro Gln Thr Phe Pro Glu Asn Ser Arg Cys Ser Trp Thr Val 2825 2830 2835 Ile Thr Gly Asp Ser Lys Gly Trp Glu Ile Ser Phe Asp Ser Asn 2840 2845 2850 Phe Arg Ile Pro Ser Ser Asp Ser Gln Cys Gln Asn Ser Phe Val 2855 2860 2865 Lys Val Trp Gly Gly Arg Leu Met Ile Asn Lys Thr Leu Leu Ala 2870 2875 2880 Thr Ser Cys Gly Asp Val Ala Pro Ser Pro Ile Val Thr Ser Gly 2885 2890 2895 Asn Ile Phe Thr Ala Val Phe Gln Ser Glu Glu Met Ala Ala Gln 2900 2905 2910 Gly Phe Ser Ala Ser Phe Ile Ser Arg Cys Gly Arg Thr Phe Asn 2915 2920 2925 Thr Ser Pro Gly Asp Ile Ile Ser Pro Asn Phe Pro Lys Gln Tyr 2930 2935 2940 Asp Asn Asn Met Asn Cys Thr Tyr Leu Ile Asp Ala Asp Pro Gln 2945 2950 2955 Ser Leu Val Ile Leu Thr Phe Val Ser Phe Gly Leu Glu Asp Arg 2960 2965 2970 Ser Ala Ile Thr Gly Thr Cys Asp Gly Asp Gly Leu Gly Ile Ile 2975 2980 2985 Lys Gly Arg Asn Leu Ser Ser Thr Pro Leu Val Thr Ile Cys Gly 2990 2995 3000 Ser Glu Thr Leu Arg Pro Leu Thr Val Asp Gly Pro Val Leu Leu 3005 3010 3015 Asn Phe Tyr Ser Asp Ala Tyr Thr Thr Asp Phe Gly Phe Lys Ile 3020 3025 3030 Ser Tyr Arg Ala Ile Thr Cys Gly Gly Ile Tyr Asn Glu Ser Ser 3035 3040 3045 Gly Ile Leu Arg Ser Pro Ser Tyr Ser Tyr Ser Asn Tyr Pro Asn 3050 3055 3060 Asn Leu Tyr Cys Val Tyr Ser Leu Gly Val Arg Ser Ser Arg Val 3065 3070 3075 Ile Ile Ile Arg Phe Asn Asp Phe Asp Val Ala Pro Ser Asn Leu 3080 3085 3090 Cys Ala Gly Asp Phe Leu Glu Val Phe Asp Gly Pro Ser Ile Gly 3095 3100 3105 Asn Arg Ser Leu Gly Lys Phe Cys Gly Ser Thr Arg Pro Gln Thr 3110 3115 3120 Val Lys Ser Thr Asn Ser Ser Leu Thr Leu Leu Phe Lys Thr Asp 3125 3130 3135 Ser Ser Gln Thr Ala Arg Gly Trp Lys Ile Phe Phe Arg Glu Thr 3140 3145 3150 Ile Gly Pro Gln Gln Gly Cys Gly Gly Tyr Leu Thr Glu Asp Asn 3155 3160 3165 Gln Ser Phe Val Ser Pro Asp Ser Asp Ser Asn Gly Arg Tyr Asp 3170 3175 3180 Lys Gly Leu Ser Cys Ile Trp Tyr Ile Val Ala Pro Glu Asn Lys 3185 3190 3195 Leu Val Lys Leu Thr Phe Asn Val Phe Thr Leu Glu Gly Pro Ser 3200 3205 3210 Ser Ala Gly Ser Cys Val Tyr Asp Tyr Val Gln Ile Ala Asp Gly 3215 3220 3225 Ala Ser Ile Asn Ser Tyr Leu Gly Gly Lys Phe Cys Gly Ser Arg 3230 3235 3240 Met Pro Ala Pro Phe Ile Ser Ser Gly Tyr Phe Leu Thr Phe Gln 3245 3250 3255 Phe Val Ser Asp Val Thr Val Glu Met Arg Gly Phe Asn Ala Thr 3260 3265 3270 Tyr Thr Phe Val Asp Met Pro Cys Gly Gly Thr Tyr Asn Ala Thr 3275 3280 3285 Ser Thr Pro Gln Asn Ala Ser Ser Pro Gly Leu Ser Asn Ile Gly 3290 3295 3300 Arg Pro Tyr Ser Thr Cys Thr Trp Val Ile Ala Ala Pro Pro Gln 3305 3310 3315 Gln Gln Val Gln Ile Thr Val Trp Asp Leu Gln Leu Pro Ser Gln 3320 3325 3330 Asp Cys Ser Gln Ser Tyr Leu Glu Leu Gln Asp Ser Val Gln Thr 3335 3340 3345 Gly Gly Asn Arg Val Thr Gln Phe Cys Gly Ala Asn Tyr Thr Thr 3350 3355 3360 Leu Pro Val Phe Tyr Ser Ser Met Ser Thr Ala Val Val Val Phe 3365 3370 3375 Lys Ser Gly Val Ile Asn Arg Asn Ser Gln Val Gln Phe Ser Tyr 3380 3385 3390 Gln Ile Ala Asp Cys Asn Arg Glu Tyr Asn Gln Thr Phe Gly Asn 3395 3400 3405 Leu Lys Ser Pro Gly Trp Pro Gln Asn Tyr Asp Asn Asn Leu Asp 3410 3415 3420 Cys Thr Ile Ile Leu Arg Ala Pro Gln Asn Gly Ser Ile Ser Leu 3425 3430 3435 Phe Phe Tyr Trp Phe Gln Leu Glu Asp Ser Arg Gln Cys Met Asn 3440 3445 3450 Asp Phe Leu Glu Val Arg Asn Gly Gly Ser Ser Thr Ser Pro Leu 3455 3460 3465 Leu Asp Lys Tyr Cys Ser Asn Leu Leu Pro Asn Pro Val Phe Ser 3470 3475 3480 Gln Ser Asn Glu Leu Tyr Leu Gly Phe Gly Ser Asp Gly Ser Val 3485 3490 3495 Thr Asn Asn Gly Tyr Glu Ile Ile Trp Thr Ser Ser Ala Ala Gly 3500 3505 3510 Cys Gly Gly Thr Leu Leu Gly Asp Glu Gly Ile Phe Thr Asn Pro 3515 3520 3525 Gly Phe Pro Asp Ser Tyr Pro Asn Asn Thr Gly Cys Glu Trp Thr 3530 3535 3540 Ile Val Ala Pro Ser Gly Arg Pro Val Ser Val Gly Phe Pro Phe 3545 3550 3555 Leu Ser Ile Asp Ser Ser Gly Gly Cys Asp Gln Asn Tyr Leu Ile 3560 3565 3570 Val Phe Asn Gly Pro Asp Ala Asn Ser Pro Pro Phe Gly Pro Leu 3575 3580 3585 Cys Gly Ile Asn Thr Gly Ile Ala Pro Phe Tyr Ala Ser Ser Asn 3590 3595 3600 Arg Val Phe Ile Arg Phe Gly Ala Glu Tyr Thr Thr Arg Leu Ser 3605 3610 3615 Gly Phe Glu Ile Met Trp Ser Ser 3620 3 22 DNA artificial sequence primer_bind bps 838-859 of rat cubilin primer for 5′ RACE 3 acacaaggct ccttctactg tg 22 4 20 DNA artificial sequence primer_bind bps 6872-6891 of rat cubilin primer for 3′ RACE 4 gtctggtttc caagttgtgt 20 5 21 DNA artificial sequence primer_bind bps 7152-7172 of rat cubilin primer for 3′ RACE 5 tcagagctct cctggttgta c 21 6 36 PRT rat amino acid sequence of rat cubilin EGF1 repeat 6 Arg Lys Val Cys Ser Ser Asn Pro Cys Leu Asn Gly Gly Thr Cys 5 10 15 Val Asn Leu His Asp Ser Phe Val Cys Ile Cys Pro Ser Gln Trp 20 25 30 Lys Gly Leu Phe Cys Ser 35 7 43 PRT rat amino acid sequence of rat cubilin EGF2 repeat 7 Glu Asp Val Asn Glu Cys Val Val Tyr Ser Gly Thr Pro Phe Gly 5 10 15 Cys Gln Ser Gly Ser Thr Cys Val Asn Thr Val Gly Ser Phe Arg 20 25 30 Cys Asp Cys Thr Pro Asp Thr Tyr Gly Pro Gln Cys Ala 35 40 8 50 PRT rat amino acid sequence of rat cubilin EGF3 repeat 8 Ser Lys Tyr Asn Asp Cys Glu Gln Gly Ser Lys Gln Leu Cys Lys 5 10 15 His Gly Ile Cys Glu Asp Leu Gln Arg Val His His Gly Gln Pro 20 25 30 Asn Phe His Cys Ile Cys Asp Ala Gly Trp Thr Thr Pro Pro Asn 35 40 45 Gly Ile Ser Cys Thr 50 9 43 PRT rat amino acid sequence of rat cubilin EGF4 repeat 9 Glu Asp Lys Asp Glu Cys Ser Leu Gln Pro Ser Pro Cys Ser Glu 5 10 15 His Ala Gln Cys Phe Asn Thr Gln Gly Ser Phe Tyr Cys Gly Ala 20 25 30 Cys Pro Lys Gly Trp Gln Gly Asn Gly Tyr Glu Cys Gln 35 40 10 44 PRT rat amino acid sequence of rat cubilin EGF5 repeat 10 Asp Ile Asn Lys Cys Glu Ile Asn Asn Gly Gly Cys Ser Gln Ala 5 10 15 Pro Leu Val Pro Cys Leu Asn Thr Pro Gly Ser Phe Ser Cys Gly 20 25 30 Asn Cys Pro Ala Gly Phe Ser Gly Asp Gly Arg Val Cys Thr 35 40 11 48 PRT rat amino acid sequence of rat cubilin EGF6 repeat 11 Pro Val Asp Ile Cys Ser Ile His Asn Gly Gly Cys His Pro Glu 5 10 15 Ala Thr Cys Ser Ser Ser Pro Val Leu Gly Ser Phe Leu Pro Val 20 25 30 Cys Thr Cys Pro Pro Gly Tyr Thr Gly Asn Gly Tyr Gly Ser Asn 35 40 45 Gly Cys Val 12 37 PRT rat amino acid sequence of rat cubilin EGF7 repeat 12 Arg Leu Ser Asn Ile Cys Ser Arg His Pro Cys Val Asn Gly Gln 5 10 15 Cys Ile Glu Thr Val Ser Ser Tyr Phe Cys Lys Cys Asp Ser Gly 20 25 30 Trp Ser Gly Gln Asn Cys Thr 35 13 38 PRT rat amino acid sequence of rat cubilin EGF8 repeat 13 Glu Asn Ile Asn Asp Cys Ser Ser Asn Pro Cys Leu Asn Gly Gly 5 10 15 Thr Cys Ile Asp Gly Ile Asn Gly Phe Thr Cys Asp Cys Thr Ser 20 25 30 Ser Trp Thr Gly Tyr Tyr Cys Gln 35 14 42 PRT human amino acid sequence of Bmp-1 EGF1 repeat 14 Glu Val Asp Glu Cys Ser Arg Pro Asn Arg Gly Gly Cys Glu Gln 5 10 15 Arg Cys Leu Asn Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro 20 25 30 Gly Tyr Glu Leu Ala Pro Asp Lys Arg Arg Cys Glu 35 40 15 41 PRT Drosophila amino acid sequence of Tolloid EGF1 repeat 15 Asp Val Asp Glu Cys Lys Phe Thr Asp His Gly Cys Gln His Leu 5 10 15 Cys Ile Asn Thr Leu Gly Ser Tyr Gln Cys Gly Cys Arg Ala Gly 20 25 30 Tyr Glu Leu Gln Ala Asn Gly Lys Thr Cys Glu 35 40 16 41 PRT Drosophila amino acid sequence of Tolloid EGF2 repeat 16 Asp Val Asp Glu Cys Ser Met Asn Asn Gly Gly Cys Gln His Arg 5 10 15 Cys Arg Asn Thr Phe Gly Ser Tyr Gln Cys Ser Cys Arg Asn Gly 20 25 30 Tyr Thr Leu Ala Glu Asn Gly His Asn Cys Thr 35 40 17 42 PRT human amino acid sequence of C1s EGF1 repeat 17 Asp Ile Asn Glu Cys Thr Asp Phe Val Asp Val Pro Cys Ser His 5 10 15 Phe Cys Asn Asn Phe Ile Gly Gly Tyr Phe Cys Ser Cys Pro Pro 20 25 30 Glu Tyr Phe Leu His Asp Asp Met Lys Asn Cys Gly 35 40 18 42 PRT human amino acid sequence of fibrillin-1 EGF5 repeat 18 Asp Ile Asp Glu Cys Ser Thr Ile Pro Gly Ile Cys Glu Gly Gly 5 10 15 Glu Cys Thr Asn Thr Val Ser Ser Tyr Phe Cys Lys Cys Pro Pro 20 25 30 Gly Phe Tyr Thr Ser Pro Asp Gly Thr Arg Cys Ile 35 40 19 41 PRT human amino acid sequence of fibrillin-1 EGF13 repeat 19 Asp Ile Asp Glu Cys Glu Ser Ser Pro Cys Ile Asn Gly Val Cys 5 10 15 Lys Asn Ser Pro Gly Ser Phe Ile Cys Glu Cys Ser Ser Glu Ser 20 25 30 Thr Leu Asp Pro Lys Thr Lys Thr Ile Cys Ile 35 40 20 41 PRT human amino acid sequence of fibrillin-1 EGF26 repeat 20 Asp Val Asn Glu Cys Leu Asp Pro Thr Thr Cys Ile Ser Gly Asn 5 10 15 Cys Val Asn Thr Pro Gly Ser Tyr Ile Cys Asp Cys Pro Pro Asp 20 25 30 Phe Glu Leu Asn Pro Thr Arg Val Gly Cys Val 35 40 21 110 PRT rat amino acid sequence of rat cubilin CUB2 domain 21 Cys Gly Gly Ile Leu Thr Asp Asn Tyr Gly Ser Ile Thr Ser Pro 5 10 15 Gly Tyr Pro Gly Asn Tyr Pro Pro Gly Arg Asp Cys Val Trp Gln 20 25 30 Val Leu Val Asn Pro Asn Ser Leu Ile Thr Phe Thr Phe Gly Thr 35 40 45 Leu Ser Leu Glu Ser His Asn Asp Cys Ser Lys Asp Tyr Leu Glu 50 55 60 Ile Arg Asp Gly Pro Phe His Gln Asp Pro Val Leu Gly Lys Phe 65 70 75 Cys Thr Ser Leu Ser Thr Pro Pro Leu Lys Thr Thr Gly Pro Ala 80 85 90 Ala Arg Ile His Gly His Ser Cys Ser Glu Thr Ser Asp Lys Gly 95 100 105 Phe His Ile Thr Tyr 110 22 108 PRT rat amino acid sequence of rat cubilin CUB5 domain 22 Cys Gly Glu Val Leu Thr Ala Ser Thr Gly Ile Ile Glu Ser Pro 5 10 15 Gly His Pro Asn Val Tyr Pro Arg Gly Val Asn Cys Thr Trp His 20 25 30 Val Val Val Gln Arg Gly Gln Leu Ile Arg Leu Glu Phe Ser Ser 35 40 45 Phe Tyr Leu Glu Phe His Tyr Asn Cys Thr Asn Asp Tyr Leu Glu 50 55 60 Ile Tyr Asp Thr Ala Ala Gln Thr Phe Leu Gly Arg Tyr Cys Gly 65 70 75 Lys Ser Ile Pro Pro Ser Leu Thr Ser Asn Ser Asn Ser Ile Lys 80 85 90 Leu Ile Phe Val Ser Asp Ser Ala Leu Ala His Glu Phe Gly Ser 95 100 105 Ile Asn Tyr 23 111 PRT rat amino acid sequence of rat cubilin CUB6 domain 23 Cys Leu Tyr Asp Tyr Thr Asp Asn Phe Gly Met Leu Ser Ser Pro 5 10 15 Asn Phe Pro Asn Asn Tyr Pro Ser Asn Trp Glu Cys Ile Tyr Arg 20 25 30 Ile Thr Val Gly Leu Asn Gln Gln Ile Ala Leu His Phe Thr Asp 35 40 45 Phe Thr Leu Glu Asp Tyr Phe Gly Ser Gln Cys Val Asp Phe Val 50 55 60 Glu Ile Arg Asp Gly Gly Tyr Glu Thr Ser Pro Leu Val Gly Ile 65 70 75 Tyr Cys Gly Ser Val Leu Pro Pro Thr Ile Ile Ser His Ser Asn 80 85 90 Lys Leu Trp Leu Lys Phe Lys Ser Asp Ala Ala Leu Thr Ala Lys 95 100 105 Gly Phe Ser Ala Tyr Trp 110 24 113 PRT rat amino acid sequence of rat cubilin CUB9 domain 24 Cys Gly Gly Glu Met Ser Gly Thr Ala Gly Ser Phe Ser Ser Pro 5 10 15 Gly Tyr Pro Asn Ser Tyr Pro His Asn Lys Glu Cys Ile Trp Asn 20 25 30 Ile Arg Val Ala Pro Gly Ser Ser Ile Gln Leu Thr Ile His Asp 35 40 45 Phe Asp Val Glu Tyr His Thr Ser Cys Asn Tyr Asp Ser Leu Glu 50 55 60 Ile Tyr Ala Gly Leu Asp Phe Asn Ser Pro Arg Ile Ala Gln Leu 65 70 75 Cys Ser Gln Ser Pro Ser Ala Asn Pro Met Gln Val Ser Ser Thr 80 85 90 Gly Asn Glu Leu Ala Ile Arg Phe Lys Thr Asp Ser Thr Leu Asn 95 100 105 Gly Arg Gly Phe Asn Ala Ser Trp 110 25 110 PRT rat amino acid sequence of rat cubilin CUB12 domain 25 Cys Gly Gly Ser Phe Tyr Thr Leu Asp Gly Ile Phe Asn Ser Pro 5 10 15 Asp Tyr Pro Ala Asp Tyr His Gly Asn Ala Glu Cys Val Trp Asn 20 25 30 Ile Ala Ser Ser Pro Gly Asn Arg Leu Gln Leu Ser Phe Leu Ser 35 40 45 Phe Asn Leu Glu Asn Ser Leu Asn Cys Asn Lys Asp Phe Val Glu 50 55 60 Ile Arg Glu Gly Asn Ala Thr Gly His Leu Ile Gly Arg Tyr Cys 65 70 75 Gly Asn Ser Leu Pro Gly Asn Tyr Ser Ser Ala Glu Gly His Ser 80 85 90 Leu Trp Val Arg Phe Val Ser Asp Gly Ser Gly Thr Gly Met Gly 95 100 105 Phe Gln Ala Arg Phe 110 26 110 PRT rat amino acid sequence of rat cubilin CUB17 domain 26 Cys Gly Gly Thr Val Ser Gly Asp Ser Gly Val Ile Glu Ser Ile 5 10 15 Gly Tyr Pro Thr Leu Pro Tyr Ala Asn Asn Val Phe Cys Gln Trp 20 25 30 Phe Ile Arg Gly Leu Pro Gly His Tyr Leu Thr Leu Ser Phe Glu 35 40 45 Asp Phe Asn Leu Gln Ser Ser Pro Gly Cys Thr Lys Asp Phe Val 50 55 60 Glu Ile Trp Glu Asn His Thr Ser Gly Arg Val Leu Gly Arg Tyr 65 70 75 Cys Gly Asn Ser Thr Pro Ser Ser Val Asp Thr Ser Ser Asn Val 80 85 90 Ala Ser Val Lys Arg Val Thr Asp Gly Ser Val Thr Ala Ser Gly 95 100 105 Phe Arg Leu Gln Phe 110 27 110 PRT rat amino acid sequence of rat cubilin CUB20 domain 27 Cys Gly Gly Ile Arg Thr Gly Asp Asn Gly Val Ile Ser Ser Pro 5 10 15 Asn Tyr Pro Asn Leu Tyr Ser Ala Trp Thr His Cys Ser Trp Leu 20 25 30 Leu Lys Ala Pro Glu Gly His Thr Ile Thr Leu Thr Leu Ser Asp 35 40 45 Phe Leu Leu Glu Ala His Pro Thr Cys Thr Ser Asp Ser Val Thr 50 55 60 Val Arg Asn Gly Asp Ser Pro Gly Ser Pro Val Ile Gly Arg Tyr 65 70 75 Cys Gly Gln Ser Val Pro Arg Pro Ile Gln Ser Gly Ser Asn Gln 80 85 90 Leu Ile Val Thr Phe Asn Thr Asn Asn Gln Gly Gln Thr Arg Gly 95 100 105 Phe Tyr Ala Thr Trp 110 28 110 PRT human amino acid sequence of Bmp-1 CUB1 domain 28 Cys Gly Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro 5 10 15 Glu Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val Trp Arg 20 25 30 Ile Ser Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser 35 40 45 Leu Asp Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu 50 55 60 Val Arg Asp Gly Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe 65 70 75 Cys Gly Ser Lys Leu Pro Glu Pro Ile Val Ser Thr Asp Ser Arg 80 85 90 Leu Trp Val Glu Phe Arg Ser Ser Ser Asn Trp Val Gly Lys Gly 95 100 105 Phe Phe Ala Val Tyr 110 29 110 PRT human amino acid sequence of Bmp-1 CUB2 domain 29 Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln Ser Pro 5 10 15 Asn Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg 20 25 30 Ile Gln Val Ser Glu Gly Phe His Val Gly Leu Thr Phe Gln Ser 35 40 45 Phe Glu Ile Glu Arg His Asp Ser Cys Ala Tyr Asp Tyr Leu Glu 50 55 60 Val Arg Asp Gly His Ser Glu Ser Ser Thr Leu Ile Gly Arg Tyr 65 70 75 Cys Gly Tyr Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg 80 85 90 Leu Trp Leu Lys Phe Val Ser Asp Gly Ser Ile Asn Lys Ala Gly 95 100 105 Phe Ala Val Asn Phe 110 30 111 PRT Drosphila amino acid sequence of Tolloid CUB2 domain 30 Cys Gly Gly Asp Leu Lys Leu Thr Lys Asp Gln Ser Ile Asp Ser 5 10 15 Pro Asn Tyr Pro Met Asp Tyr Met Pro Asp Lys Glu Cys Val Trp 20 25 30 Arg Ile Thr Ala Ala Pro Asp Asn His Gln Val Ala Leu Lys Phe 35 40 45 Gln Ser Phe Glu Leu Glu Lys His Asp Gly Cys Ala Tyr Asp Phe 50 55 60 Val Glu Ile Arg Asp Gly Asn His Ser Asp Ser Arg Leu Ile Gly 65 70 75 Arg Phe Cys Gly Lys Leu Pro Pro Asn Ile Lys Thr Arg Ser Asn 80 85 90 Gln Met Tyr Ile Arg Phe Val Ser Asp Ser Ser Val Gln Lys Leu 95 100 105 Gly Phe Ser Ala Ala Leu 110 31 116 PRT Drosphila amino acid sequence of Tolloid CUB3 domain 31 Cys Gly Gly Val Val Asp Ala Thr Lys Ser Asn Gly Ser Leu Tyr 5 10 15 Ser Pro Ser Tyr Pro Asp Val Tyr Pro Asn Ser Lys Gln Cys Val 20 25 30 Trp Glu Val Val Ala Pro Pro Asn His Ala Val Phe Leu Asn Phe 35 40 45 Ser His Phe Asp Leu Glu Gly Thr Arg Phe His Tyr Thr Lys Cys 50 55 60 Asn Tyr Asp Tyr Leu Ile Ile Tyr Ser Lys Met Arg Asp Asn Arg 65 70 75 Leu Lys Lys Ile Gly Ile Tyr Cys Gly His Glu Leu Pro Pro Val 80 85 90 Val Asn Ser Glu Gln Ser Ile Leu Arg Leu Glu Phe Tyr Ser Asp 95 100 105 Arg Thr Val Gln Arg Ser Gly Phe Val Lys Phe 110 115 32 109 PRT Drosphila amino acid sequence of Tolloid CUB4 domain 32 Cys Lys Phe Glu Ile Thr Thr Ser Tyr Gly Val Leu Gln Ser Pro 5 10 15 Asn Tyr Pro Glu Asp Tyr Pro Arg Asn Ile Tyr Cys Tyr Trp His 20 25 30 Phe Gln Thr Val Leu Gly Phe Ile Gln Leu Thr Phe His Asp Phe 35 40 45 Glu Val Glu Ser His Gln Glu Cys Ile Tyr Asp Tyr Val Ala Ile 50 55 60 Tyr Asp Gly Arg Ser Glu Asn Ser Ser Thr Leu Gly Ile Tyr Cys 65 70 75 Gly Gly Arg Glu Pro Tyr Ala Val Ile Ala Ser Thr Asn Glu Met 80 85 90 Phe Met Val Leu Ala Thr Asp Ala Gly Leu Gln Arg Lys Gly Phe 95 100 105 Lys Ala Thr Phe 33 109 PRT Xenopus laevis amino acid sequence of Uvs-2 CUB2 domain 33 Cys Gly Gly Ala Phe Tyr Ser Ser Pro Lys Thr Phe Thr Ser Pro 5 10 15 Asn Tyr Pro Gly Asn Tyr Thr Thr Asn Thr Asn Cys Thr Trp Thr 20 25 30 Ile Thr Ala Pro Ala Gly Phe Lys Val Ser Leu Arg Ile Thr Asp 35 40 45 Phe Glu Leu Glu Ile Gly Ala Ser Cys Arg Tyr Asp Tyr Leu Asn 50 55 60 Ile Tyr Asn Ser Thr Leu Gly Ala Val Met Gly Pro Tyr Cys Gly 65 70 75 Pro Ile Asp Phe His Ser Ala Ile Val Ser Lys Ser Asn Ser Met 80 85 90 Met Ile Thr Met Asn Ser Asp Phe Ser Lys Gln Tyr Lys Gly Phe 95 100 105 Ser Ala Thr Tyr 34 112 PRT human amino acid sequence of C1s CUB1 domain 34 Glu Pro Thr Met Tyr Gly Glu Ile Leu Ser Pro Asn Tyr Pro Gln 5 10 15 Ala Tyr Pro Ser Glu Val Glu Lys Ser Trp Asp Ile Glu Val Pro 20 25 30 Glu Gly Tyr Gly Ile His Leu Tyr Phe Thr His Leu Asp Ile Glu 35 40 45 Leu Ser Glu Asn Cys Ala Tyr Asp Ser Val Gln Ile Ile Ser Gly 50 55 60 Asp Thr Glu Glu Gly Arg Leu Cys Gly Gln Arg Ser Ser Asn Asn 65 70 75 Pro His Ser Pro Ile Val Glu Glu Phe Gln Val Pro Tyr Asn Lys 80 85 90 Leu Gln Val Ile Phe Lys Ser Asp Phe Ser Asn Glu Glu Arg Phe 95 100 105 Thr Gly Phe Ala Ala Tyr Tyr 110 35 109 PRT human amino acid sequence of Tsg6 CUB domain 35 Cys Gly Gly Val Gly Thr Asp Pro Lys Arg Ile Phe Lys Ser Pro 5 10 15 Gly Phe Pro Asn Glu Tyr Glu Asp Asn Gln Ile Cys Tyr Trp His 20 25 30 Ile Arg Leu Lys Tyr Gly Gln Arg Ile His Leu Ser Phe Leu Asp 35 40 45 Phe Asp Leu Glu Asp Asp Pro Gly Cys Leu Ala Asp Tyr Val Glu 50 55 60 Ile Tyr Asp Ser Tyr Asp Asp Val His Gly Phe Val Gly Arg Tyr 65 70 75 Cys Gly Asp Glu Leu Pro Asp Asp Ile Ile Ser Gly Asn Val Met 80 85 90 Thr Leu Lys Phe Leu Ser Asp Ala Ser Val Thr Ala Gly Gly Phe 95 100 105 Gln Ile Lys Tyr 36 98 PRT pig amino acid sequence of Aqn-3 CUB domain 36 Cys Gly Gly Phe Leu Lys Asn Tyr Ser Gly Trp Ile Ser Tyr Tyr 5 10 15 Lys Ala Leu Thr Thr Asn Cys Val Trp Thr Ile Glu Met Lys Pro 20 25 30 Gly His Lys Ile Ile Leu Gln Ile Leu Pro Leu Asn Leu Thr Cys 35 40 45 Lys Glu Tyr Leu Glu Val Arg Asp Gln Arg Ala Gly Pro Asp Asn 50 55 60 Phe Leu Lys Val Cys Gly Gly Thr Gly Phe Val Tyr Gln Ser Ser 65 70 75 His Asn Val Ala Thr Val Lys Tyr Ser Arg Asp Ser His His Pro 80 85 90 Ala Ser Ser Phe Asn Val Tyr Phe 95 37 21 DNA artificial sequence primer_bind a cubilin primer used for RT-PCR 37 tgcctaccac agcccaaatg a 21 38 19 DNA artificial sequence primer_bind a cubilin primer used for RT-PCR 38 agagccacaa tgactgcag 19 39 22 DNA artificial sequence primer_bind a megalin primer used for RT-PCR 39 gccagggaga caggaacagt ag 22 40 22 DNA artificial sequence primer_bind a megalin primer used for RT-PCR 40 tcacaaaatg ccagaccacg aa 22 

What is claimed is:
 1. An isolated and purified cubilin protein, where said protein has an amino acid sequence shown in SEQ ID No. 2, and where said protein is encoded by DNA selected from the group consisting of: (a) an isolated DNA which encodes a cubilin protein; and (b) an isolated DNA differing from the isolated DNA of (a) in codon sequence due to the degeneracy of the genetic code.
 2. A composition comprising the protein or ligand binding fragment of claim 1 and a pharmaceutically acceptable carrier.
 3. A receptor for ligands, where said receptor is the cubilin protein of claim 1 and where said receptor comprises a cluster of EGF repeats and a cluster of CUB domains.
 4. The receptor of claim 3, wherein said ligand is selected from the group consisting of immunoglobulin light chain, myoglobin, intrinsic factor-vitamin B₁₂, metallothionein, β-2-microglobulin, amyloid, hemoglobin, haptoglobin, interferon, insulin, cytochrome c, lysozyme, transferrin, transthyretin, polybasic drugs, apolipoprotein Al, high density lipoprotein and receptor related protein.
 5. The receptor of claim 4, wherein said immunoglobulin light chain is selected from the group consisting of κ-light chain and λ-light chain. 